Chromosomal, microsatellite and mitochondrial DNA variability of Drosophila subobscura population from gorges and canyons in Serbia

Drosophila subobscura je vrsta sa širokim Palearktičkim rasprostranjenjem. Naseljava gotovo ceo evropski kontinent, severnu Afriku, Malu Aziju i neka atlanska ostrva. Genetička struktura prirodnih populacija ove vrste je opisana upotrebom nekoliko genetičkih markera. Najtemeljnije je izučena varijabilnost hromozomskog inverzionog polimorfizma, po kome populacije D. subobscura pokazuju prostornu i vremensku struktuiranost. Klinalni obrasci ovog tipa varijabilnosti upućuju na značaj selektivnih procesa u njenom oblikovanju, dok razlike između geografski bliskih područja između kojih postoji geografska barijera upućuju na značaj istorijskih procesa. Naime, uočena varijabilnost oslikava procese rekolonizacije Evrope nakon završetka poslednje glacijacije iz primarnog pribežišta lociranog u regionu koji obuhvata Balkan, Malu Aziju i Kavkaz, ali i sekundarnog u regionu Pirinejskog poluostrva. Mikrosatelitska varijabilnost izučavana je u nekoliko evropskih populacija D. subobscura, uglavnom iz zapadnog dela Evrope. Pokazano je da su populacije slabo struktuirane po ovom genetičkom markeru. Populacije u južnom delu areala odlikuju se nešto većom genetičkom raznovrsnošću. Donekle je slična i slika varijabilnosti prirodnih populacija po mitohondrijalnoj DNK (mtDNK), u slučaju koje je zabeležena homogenost distribucije haplotipova između populacija. U velikoj većini do sada analiziranih populacija zabeleženo je prisustvo dva dominantna haplotipa i veliki broj endemičnih haplotipova niske učestalosti. Pokazano je da nekoliko faktora oblikuje varijabilnost mtDNK D.subobscura. Među njima su najznačajniji istorijski procesi, genetički drift, i prirodna selekcija koja najverovatnije deluje preko cito-nukleusne koadaptacije između alelskih kombinacija u okviru inverzija i mtDNK varijanti...Drosophila subobscura shows a wide Palearctic distribution. It inhabits most of Europe, Northern Africa, Asia Minor and some Atlantic islands. The genetic structure in natural populations of this species has been extensively studied with respect to several genetic markers. The variability of chromosomal inversion polymorphism has been most thoroughly studied showing spatial and temporal structure in natural populations. The clinal pattern of chromosomal variability suggests the action of natural selection on inversion polymorphism, while abrupt changes in gene arrangement frequencies between closely related populations separated by geographical barrier suggest the importance of historical processes on the genetic structure. After the decay of the ice caps, Drosophila subobscura, most probably, recolonized Europe mainly from the Balkan Peninsula, Asia Minor and Caucasus, but also, to a lesser degree, from the Iberian Peninsula. Several populations of D. subobscura, mostly from the Western Europe, have been studied with respect to microsatellite variability. The analysis showed absence of structuring of genetic variation, with slightly higher genetic variability in Southern populations. There is a widespread genetic homogeneity of mitochondrial DNA (mtDNA) variability throughout the species range with high prevalence of two almost equally frequent haplotypes, and a number of population specific, rare haplotypes. Several factors account for the observed distribution in mtDNA variability: historical processes, genetic drift and natural selection, most probably acting through cytonuclear coadaptation of alleles inside inversions and mtDNA haplotypes..

Relationship between chromosomal and mitochondrial DNA variability of Drosophila subobscura population from the Lazar's river canyon

The genetic structure of Drosophila subobscura population from the Lazar’s River Canyon (Serbia) was studied with respect to restriction site polymorphism of mitochondrial DNA and chromosomal inversion polymorphism. The aim was to shed more light on the role of cytonuclear interactions in shaping mitochondrial DNA variability in this species. Similar to other populations of D. subobscura two main haplotypes (I and II) were found, as well as less common ones that appeared at very low frequencies. The frequency distribution of haplotypes did not depart from neutrality. We did not find statistically significant linkage disequilibrium between the haplotypes belonging to haplogroups I and II and any of the chromosomal arrangements. However, when we compared the data in hereby analyzed population and two previously analyzed populations we observed that haplotype I is more frequent in populations where standard inversion arrangements are less frequent. Pattern of the observed mitochondrial variability could be influenced either directly by environmental variability or through environmentally specific cytonuclear coadaptation.U ovom radu analizirana je genetička varijabilnost restrikcionih mesta mitohondrijalne DNK, kao i varijabilnost inverzionog polimorfizma u populaciji Drosophila subobscura sakupljenoj u kanjonu Lazareve reke. Cilj je bio rasvetljavanje uloge citonuklearnih interakcija u oblikovanju varijabilnosti mitohondrijalne DNK. Utvrđeno je prisustvo dva dominantna haplotipa (I i II) kao i dva haplotipa niske učestalosti. Nije utvrđena statistički značajna neravnoteža vezanosti između haplotipova grupisanih u haplogrupe I i II i nijednog od hromozomskih aranžmana. Međutim, upoređivanjem podataka prethodno analiziranih populacija i populacije analizirane u ovom radu, primećuje se povećanje učestalosti haplotipa I sa smanjenjem učestalosti standardnih hromozomskih aranžmana. Ovakav obrazac ukazuje ili na direktan uticaj sredinskih činilaca na varijabilnost mitohondrijalne DNK ili na sredinski zavisnu citonuklearnu koadaptaciju.Projekat ministarstva br. 17301

Sexual selection can reduce mutational load in Drosophila subobscura

According to theoretical predictions sexual selection can reduce mutational load through male mating success. Males of good genetic quality should be more successful in matings, compared to the males of low genetic quality, thus in this way females can prevent deleterious alleles to be transmitted to the next generation. We tested this hypothesis through set up of two experimental groups from same genetic pool, where in one group genetic quality was manipulated by ionizing radiation. Within each group opportunity for choosing mates was imposed: males and females had no choice or had multiple choice. Mutational load was measured through the variability of different fitness components: fecundity and egg-to-adult viability. Our results indicate that sexual selection can reduce mutational load, only for fecundity. Group with the presence of female choice exhibited higher fecundity than group in which sexual selection was experimentally eliminated, but only in 'irradiated' group. There was no overall difference in egg-to-adult viability between different sexual selection regimes in any of the group. It should be considered that sexual selection can cause sexual conflict, and potential opposite effects of sexual selection and sexual conflict on fitness. Genetic structure of populations, in terms of the level of mutational load, is an important factor which can determinate the role of sexual selection.Prema teorijskim očekivanjima seksualna selekcija može smanjivati mutaciona opterećenja u populacijama, preko uspešnosti mužjaka u parenju. Mužjaci koji su boljeg genetičkog kvaliteta bi trebalo da budu uspešniji u parenju od mužjaka lošijeg genetičkog kvaliteta. Na taj način bi ženke mogle da smanje prenošenje štetnih alela u sledeću generaciju. Ova hipoteza je testirana uspostavljanjem dve eksperimentalne grupe od istog genetičkog pula jedinki, pri čemu su u jednoj mutacije indukovane jonizujućim zračenjem. Unutar svake grupe je nametnuta mogućnost izbora u parenju: mužjaci i ženke nisu imali izbor, ili su imali višestruki izbor. Mutaciona opterećenja su merena preko dve komponente adaptivne vrednosti: fekunditeta i preživljavanja od stadijuma jaja do adulta. Dobijeni rezultati ukazuju da seksualna selekcija može imati uticaj na smanjivanje mutacionih opterećenja ali samo za fekunditet. U prisustvu seksualne selekcije su vrednosti fekunditeta bile veće u odnosu na vrednosti koje su dobijene kad je seksualna selekcija eksperimentalno uklonjena, ali samo u okviru 'ozračene' grupe. Razlike u preživljavanju od jaja do adulta između različitih režima seksualne selekcije nisu dobijene ni u jednoj od grupa. Kako seksualna selekcija često uzrokuje i seksualni konflikt, treba uzeti u obzir njihovo potencijalno delovanje u suprotnim pravcima na adaptivnu vrednost. Genetička struktura populacija, koja podrazumeva i veličinu mutacionih opterećenja, može biti važan faktor od koga će i zavisiti delovanje seksualne selekcija na adaptivnu vrednost.Projekat ministarstva br. 17301

Relationship between chromosomal and mitochondrial DNA variability of Drosophila subobscura population from the Lazar's river canyon

The genetic structure of Drosophila subobscura population from the Lazar’s River Canyon (Serbia) was studied with respect to restriction site polymorphism of mitochondrial DNA and chromosomal inversion polymorphism. The aim was to shed more light on the role of cytonuclear interactions in shaping mitochondrial DNA variability in this species. Similar to other populations of D. subobscura two main haplotypes (I and II) were found, as well as less common ones that appeared at very low frequencies. The frequency distribution of haplotypes did not depart from neutrality. We did not find statistically significant linkage disequilibrium between the haplotypes belonging to haplogroups I and II and any of the chromosomal arrangements. However, when we compared the data in hereby analyzed population and two previously analyzed populations we observed that haplotype I is more frequent in populations where standard inversion arrangements are less frequent. Pattern of the observed mitochondrial variability could be influenced either directly by environmental variability or through environmentally specific cytonuclear coadaptation.U ovom radu analizirana je genetička varijabilnost restrikcionih mesta mitohondrijalne DNK, kao i varijabilnost inverzionog polimorfizma u populaciji Drosophila subobscura sakupljenoj u kanjonu Lazareve reke. Cilj je bio rasvetljavanje uloge citonuklearnih interakcija u oblikovanju varijabilnosti mitohondrijalne DNK. Utvrđeno je prisustvo dva dominantna haplotipa (I i II) kao i dva haplotipa niske učestalosti. Nije utvrđena statistički značajna neravnoteža vezanosti između haplotipova grupisanih u haplogrupe I i II i nijednog od hromozomskih aranžmana. Međutim, upoređivanjem podataka prethodno analiziranih populacija i populacije analizirane u ovom radu, primećuje se povećanje učestalosti haplotipa I sa smanjenjem učestalosti standardnih hromozomskih aranžmana. Ovakav obrazac ukazuje ili na direktan uticaj sredinskih činilaca na varijabilnost mitohondrijalne DNK ili na sredinski zavisnu citonuklearnu koadaptaciju.Projekat ministarstva br. 17301

Local adaptation at fine spatial scale through chromosomal inversions and mito-nuclear epistasis: Findings in Drosophila subobscura (Diptera: Drosophilidae)

To explore local adaptation in wild populations at a fine spatial scale we characterized the genetic variability of eight closely located populations of Drosophila subobscura and its associations with microhabitat environmental conditions. Three different genetic markers were assessed: chromosomal inversions, a SNP of mitochondrial ND5 gene and nuclear microsatellites. Population genetic analyses of chromosomal variability revealed significant genetic differentiation between these populations. Gene arrangement frequencies on the E chromosome contributed most to these differences. We also investigated role of mito-nuclear epistasis in mitochondrial genome differentiation and revealed weak linkage disequilibrium (LD) exclusively between O3+4 inversion arrangement and mitochondrial DNA haplotype I in two populations. In addition, the trend in the LD between OST chromosomal arrangement and haplotype II was general in the total sample. Microsatellite analysis revealed an absence of stochastic processes, like census reduction, upon population differentiation. Only a small amount of the genetic variation is related to geographic distance, while most (97%) is attributable to other factors and in some degree to microhabitat variables (temperature, humidity). The analysis of these factors revealed they effect inversion arrangement frequencies, especially E1+2+9, EST and OST. Even though this model organism is known for its high mobility and mostly large effective population size, the results presented here reveal that local adaptations can occur even at a small spatial scale. We propose that locally adapted alleles within chromosomal inversions, as well as joint selective pressures acting on mitochondrial and nuclear genomes, are responsible for the observed adaptation to microhabitat conditions

Nucleotide diversity of Cyt b gene in drosophila subobscura Collin

Mitochondrial DNA variability of Drosophila subobscura Collin from Southeastern Serbia was studied with respect to Restriction Site Analysis (RSA) of complete mitochondrial genome and the nucleotide sequence of Cytochrome b (Cyt b) gene. The aim was to shed more light on the evolutionary forces that shape mtDNA variation of this species. Samples were collected from two sites in the foothills of the Balkan Mountains. No genetic differentiation was found between groups and most of the variation was observed within them. Restriction analysis revealed two main haplotypes and several rare ones. The sequencing of Cyt b gene showed larger number of haplotypes, among which, one is being the most common. The majority of singletons differed from the most frequent haplotype by one nucleotide change. Although some of the observed nucleotide differences may affect their host’s fitness, the observed pattern of variation is consistent with the seasonal fluctuations in population size

Sexual selection can reduce mutational load in Drosophila subobscura

According to theoretical predictions sexual selection can reduce mutational load through male mating success. Males of good genetic quality should be more successful in matings, compared to the males of low genetic quality, thus in this way females can prevent deleterious alleles to be transmitted to the next generation. We tested this hypothesis through set up of two experimental groups from same genetic pool, where in one group genetic quality was manipulated by ionizing radiation. Within each group opportunity for choosing mates was imposed: males and females had no choice or had multiple choice. Mutational load was measured through the variability of different fitness components: fecundity and egg-to-adult viability. Our results indicate that sexual selection can reduce mutational load, only for fecundity. Group with the presence of female choice exhibited higher fecundity than group in which sexual selection was experimentally eliminated, but only in 'irradiated' group. There was no overall difference in egg-to-adult viability between different sexual selection regimes in any of the group. It should be considered that sexual selection can cause sexual conflict, and potential opposite effects of sexual selection and sexual conflict on fitness. Genetic structure of populations, in terms of the level of mutational load, is an important factor which can determinate the role of sexual selection.Prema teorijskim očekivanjima seksualna selekcija može smanjivati mutaciona opterećenja u populacijama, preko uspešnosti mužjaka u parenju. Mužjaci koji su boljeg genetičkog kvaliteta bi trebalo da budu uspešniji u parenju od mužjaka lošijeg genetičkog kvaliteta. Na taj način bi ženke mogle da smanje prenošenje štetnih alela u sledeću generaciju. Ova hipoteza je testirana uspostavljanjem dve eksperimentalne grupe od istog genetičkog pula jedinki, pri čemu su u jednoj mutacije indukovane jonizujućim zračenjem. Unutar svake grupe je nametnuta mogućnost izbora u parenju: mužjaci i ženke nisu imali izbor, ili su imali višestruki izbor. Mutaciona opterećenja su merena preko dve komponente adaptivne vrednosti: fekunditeta i preživljavanja od stadijuma jaja do adulta. Dobijeni rezultati ukazuju da seksualna selekcija može imati uticaj na smanjivanje mutacionih opterećenja ali samo za fekunditet. U prisustvu seksualne selekcije su vrednosti fekunditeta bile veće u odnosu na vrednosti koje su dobijene kad je seksualna selekcija eksperimentalno uklonjena, ali samo u okviru 'ozračene' grupe. Razlike u preživljavanju od jaja do adulta između različitih režima seksualne selekcije nisu dobijene ni u jednoj od grupa. Kako seksualna selekcija često uzrokuje i seksualni konflikt, treba uzeti u obzir njihovo potencijalno delovanje u suprotnim pravcima na adaptivnu vrednost. Genetička struktura populacija, koja podrazumeva i veličinu mutacionih opterećenja, može biti važan faktor od koga će i zavisiti delovanje seksualne selekcija na adaptivnu vrednost.Projekat ministarstva br. 17301

A Rapid and Cost-effective Procedure for Delineation and Utilization of Genomic Microsatellites for Paralleled Genotyping in Vicia faba

Although more than 400 microsatellite loci are currently available for Vicia faba L. (faba bean), an important food and feed grain crop legume, they have not yet been used for comprehensive molecular characterization of this crop. We report a three-step procedure for rapid and cost-effective delineation and utilization of informative genomic nuclear SSRs for paralleled genotyping in faba bean suitable also for other species: (i) pre-selection of loci generating PCR products of expected lengths which are potentially polymorphic (achieved by PCR amplification in bulked samples); (ii) exclusion of loci burdened with persistent null alleles and multilocus amplification products (based on PCR amplification of pre-selected loci in individual genotypes), and (iii) multiplexing. We demonstrate also that genomic SSRs are promising molecular tools for molecular characterization of faba bean required also for crop improvement

Pursuit for est microsatellites in a tetraploid model from de novo transcriptome sequencing

Available scientific literature reports very few microsatellite markers derived from tetraploid genomes using de novo transcriptome sequencing, mostly because their gain usually represents a major computational challenge due to complicated combinatorics during assembly of sequence reads. Here we present a novel approach for mining polymorphic microsatellite loci from transcriptome data in a tetraploid species with no reference genome available. Pairs of 114 bp long de novo sequenced transcriptome reads of Centaurium erythraea were merged into short contigs of 170-200 bp each. High accuracy assembly of the pairs of reads was accomplished by a minimum of 14 bp overlap. Sequential bioinformatics operations involved fully free and open-source software and were performed using an average personal computer. Out of the 13 150 candidate contigs harboring SSR motifs obtained in a final output, we randomly chose 16 putative markers for which we designed primers. We tested the effectiveness of the established bioinformatics approach by amplifying them in eight different taxa within the genus Centaurium having various ploidy levels (diploids, tetraploids and hexaploids). Nine markers displayed polymorphism and/or transferability among studied taxa. They provided 54 alleles in total, ranging from 2 to 14 alleles per locus. The highest number of alleles was observed in C. erythraea, C. littorale and a hybridogenic taxon C. pannonicum. The developed markers are qualified to be used in genetic population studies on declining natural populations of Centaurium species, thus providing valuable information to evolutionary and conservation biologists. The developed cost-effective methodology provides abundant de novo assembled short contigs and holds great promise to mine numerous additional EST-SSR-containing markers for possible use in genetics population studies of tetraploid taxa within the genus Centauriu

Phenotypic and Genetic Variation of an Interspecific Centaurium Hybrid (Gentianaceae) and Its Parental Species.

Interspecific hybridization is one of the major actuators of evolutionary changes in plants. As the result of allopolyploid hybridization, offspring may gain different ploidy levels in comparison to parental species, which can provide them instant reproductive isolation. Two tetraploid sister species, Centaurium erythraea and C. littorale, readily cross-fertilize, resulting in hybrids of various ploidy. In northern Serbia, two stable populations of a hexaploid taxon C. pannonicum have been documented. It has been proposed previously that this taxon emerged after an interspecific hybridization event between two tetraploid sister-species: C. erythraea and C. littorale subsp. compressum. The existing populations of the hybridogenic taxon, as well as neighboring populations of the two parental taxa were here characterized by both morphometrics and molecular markers (EST-SSR and trnL-F). Three leaf and two flower characteristics were found to be informative in delimitation of the parental taxa and in their discernment from hybrid individuals, the latter having intermediate values. Eight microsatellite markers were found to have good ability to distinguish studied taxa, placing C. pannonicum in closer relationship with C. erythraea. Conversely, trnL-F plastid marker nominated C. littorale subsp. compressum to be the donor of the C. pannonicum plastid DNA. Reproductive isolation of the hexaploid hybrid individuals from the parental species should be examined as the next logical step in describing the new species