Metin Tuna

Development of SSR markers and analysis of diversity in Turkish populations of Brachypodium distachyon

BackgroundBrachypodium distachyon (Brachypodium) is rapidly emerging as a powerful model system to facilitate research aimed at improving grass crops for grain, forage and energy production. To characterize the natural diversity of Brachypodium and provide a valuable new tool to the growing list of resources available to Brachypodium researchers, we created and characterized a large, diverse collection of inbred lines.ResultsWe developed 84 inbred lines from eight locations in Turkey. To enable genotypic characterization of this collection, we created 398 SSR markers from BAC end and EST sequences. An analysis of 187 diploid lines from 56 locations with 43 SSR markers showed considerable genotypic diversity. There was some correlation between SSR genotypes and broad geographic regions, but there was also a high level of genotypic diversity at individual locations. Phenotypic analysis of this new germplasm resource revealed considerable variation in flowering time, seed size, and plant architecture. The inbreeding nature of Brachypodium was confirmed by an extremely high level of homozygosity in wild plants and a lack of cross-pollination under laboratory conditions.ConclusionTaken together, the inbreeding nature and genotypic diversity observed at individual locations suggest a significant amount of long-distance seed dispersal. The resources developed in this study are freely available to the research community and will facilitate experimental applications based on natural diversity.

Genome diversity in Brachypodium distachyon: deep sequencing of highly diverse inbred lines

Brachypodium distachyon is small annual grass that has been adopted as a model for the grasses. Its small genome, high-quality reference genome, large germplasm collection, and selfing nature make it an excellent subject for studies of natural variation. We sequenced six divergent lines to identify a comprehensive set of polymorphisms and analyze their distribution and concordance with gene expression. Multiple methods and controls were utilized to identify polymorphisms and validate their quality. mRNA-Seq experiments under control and simulated drought-stress conditions, identified 300 genes with a genotype-dependent treatment response. We showed that large-scale sequence variants had extremely high concordance with altered expression of hundreds of genes, including many with genotype-dependent treatment responses. We generated a deep mRNA-Seq dataset for the most divergent line and created a de novo transcriptome assembly. This led to the discovery of >2400 previously unannotated transcripts and hundreds of genes not present in the reference genome. We built a public database for visualization and investigation of sequence variants among these widely used inbred lines.

Orchardgrass (Dactylis glomerata L.) EST and SSR marker development, annotation, and transferability.

Orchardgrass, or cocksfoot [Dactylis glomerata (L.)], has been naturalized on nearly every continent and is a commonly used species for forage and hay production. All major cultivated varieties of orchardgrass are autotetraploid, and few tools or information are available for functional and comparative genetic analyses and improvement of the species. To improve the genetic resources for orchardgrass, we have developed an EST library and SSR markers from salt, drought, and cold stressed tissues. The ESTs were bi-directionally sequenced from clones and combined into 17,373 unigenes. Unigenes were annotated based on putative orthology to genes from rice, Triticeae grasses, other Poaceae, Arabidopsis, and the non-redundant database of the NCBI. Of 1,162 SSR markers developed, approximately 80% showed amplification products across a set of orchardgrass germplasm, and 40% across related Festuca and Lolium species. When orchardgrass subspecies were genotyped using 33 SSR markers their within-accession similarity values ranged from 0.44 to 0.71, with Mediterranean accessions having a higher similarity. The total number of genotyped bands was greater for tetraploid accessions compared to diploid accessions. Clustering analysis indicated grouping of Mediterranean subspecies and central Asian subspecies, while the D. glomerata ssp. aschersoniana was closest related to three cultivated varieties.

Genome size variation in diploid and tetraploid wild wheats.

Low but significant intraspecific genome size variations were found in diploid and tetraploid wild wheats. This limited variation is not correlated with geographical and climate variables. It can be concluded that the genome size of Triticum species is generally stable, despite of the presence of many potentially active retroelements.

Diploid Brachypodium distachyon of Turkey: Molecular and Morphologic Analysis

Brachypodium distachyon is a model species for the grass family, Poaceae, which includes major cereal crops such as wheat and barley. The aim of this study were to assess morphological and phylogenetic relationships among diploid accessions of Brachypodium representing diverse geographic regions of Turkey based on Sequence related Amplified Polymorphism (SRAP) analyses. The similarity matrix indicated close relation among species used in the section using SRAP primer combinations, produced 156 fragment bands, of which 120 were polymorphic. Genetic distance ranged from 0.03 to 0.62. Plant genotypes were grouped into two major clusters based on SRAP analysis. There was a high level of diversity among the native diploid Brachypodium genotypes. These genotypes can be used for a better understanding of grass genomics.

Targeting environmental adaptation in the monocot model Brachypodium distachyon: a multi-faceted approach.

BackgroundThe local environment plays a major role in the spatial distribution of plant populations. Natural plant populations have an extremely poor displacing capacity, so their continued survival in a given environment depends on how well they adapt to local pedoclimatic conditions. Genomic tools can be used to identify adaptive traits at a DNA level and to further our understanding of evolutionary processes. Here we report the use of genotyping-by-sequencing on local groups of the sequenced monocot model species Brachypodium distachyon. Exploiting population genetics, landscape genomics and genome wide association studies, we evaluate B. distachyon role as a natural probe for identifying genomic loci involved in environmental adaptation.ResultsBrachypodium distachyon individuals were sampled in nine locations with different ecologies and characterized with 16,697 SNPs. Variations in sequencing depth showed consistent patterns at 8,072 genomic bins, which were significantly enriched in transposable elements. We investigated the structuration and diversity of this collection, and exploited climatic data to identify loci with adaptive significance through i) two different approaches for genome wide association analyses considering climatic variation, ii) an outlier loci approach, and iii) a canonical correlation analysis on differentially sequenced bins. A linkage disequilibrium-corrected Bonferroni method was applied to filter associations. The two association methods jointly identified a set of 15 genes significantly related to environmental adaptation. The outlier loci approach revealed that 5.7% of the loci analysed were under selection. The canonical correlation analysis showed that the distribution of some differentially sequenced regions was associated to environmental variation.ConclusionsWe show that the multi-faceted approach used here targeted different components of B. distachyon adaptive variation, and may lead to the discovery of genes related to environmental adaptation in natural populations. Its application to a model species with a fully sequenced genome is a modular strategy that enables the stratification of biological material and thus improves our knowledge of the functional loci determining adaptation in near-crop species. When coupled with population genetics and measures of genomic structuration, methods coming from genome wide association studies may lead to the exploitation of model species as natural probes to identify loci related to environmental adaptation.

Karyomorphology and nuclear DNA content of sixteen Ophrys L. taxa from Turkey

Abstract Karyotypes in 16 representative taxa of the Ophrys genus are compared, based on Feulgen-stained somatic metaphase chromosomes. The karyotypes of O. omegaifera subsp. israelitica, O. ulupinara, O. lycia, O. argolica subsp. lucis, O. argolica subsp. lesbis, O. climacis and O. reinholdii subsp. reinholdii are described for the first time. Karyological analyses indicate relationships among the species with respect to their asymmetry indices. All Ophrys taxa studied were diploid with 2n = 2x = 36 chromosomes. One B chromosome has been detected among the chromosomes of O. argolica subsp. lucis. All karyotypes are symmetrical, consisting of metacentric and submetacentric chromosomes. The longest chromosomes of all the investigated specimens contain a secondary constriction. It is determined that there is a correlation between the total number of chromosomes having secondary constrictions and the evolutionary development order of the taxa. Based on nuclear DNA content, analysis was carried out by flow cytometer using propodium iodide as fluorochrome, 2C nuclear DNA content of 16 Ophrys species varying between 20.80 pg (O. argolica subsp. lucis) and 23.11 pg (O. omegaifera subsp. israelitica). Karyotype asymmetry relationships are discussed according to the bidimensional scatter plots of A1–A2, CVCL–CVCI, CVCL–MCA and CVCI–MCA.

Flow Sitometri ile Çok Yıllık Buğdaygil Yem Bitkisi Genetik Kaynaklarının Karakterizasyonu

Cok yillik bugdaygil yem bitkisi turlerinin morfolojik olarak birbirlerine cok benzemeleri, aralarinda kolayca melezlenerek hibrit turler olusturabilmeleri ve dogal varyasyon sebebiyle teshislerinde ciddi sorunlar yasanmaktadir. Buna ilave olarak bu turlerde polyploidi cok yaygindir ve ayni turun dahi farkli kromozom sayilarina sahip formlari mevcuttur. Bundan dolayi cok yillik bugdaygil turlerine ait genetik kaynaklarin bilimsel arastirma ve islah calismalarinda kullanilmadan once tur teshislerinin dogru bir sekilde yapilarak ploidi duzeylerinin belirlenmesi zorunludur. Aksi taktirde yapilacak olan melezlemelerde ortaya cikabilecek genetik uyusmazlik ve kisirlik gibi sorunlar arastiricilarin zaten kit olan emek, zaman ve maddi kaynaklarinin heba olmasina sebep olmaktadir. Bu calismada, islah programlarinda kullanmak amaciyla Dogu Anadolu Bolgesi daglik bolgelerinden toplanmis olan 169 bugdaygil yem bitkisi populasyonunun ( Festuca sp., Koeleria sp. ve Agropyron sp.) cekirdek DNA icerikleri flow sitometri yontemi ile ilk defa belirlenmis ve populasyonlarin ploidi duzeyi ile safiyetlerinin belirlenmesinde kullanilmistir. Yapilan cekirdek DNA analizi sonuclarina gore Festuca populasyonlarinda ploidy duzeyi diploid ile octoploid (2n=14, 28, 42, ve 56) arasinda degisirken Koeleria populasyonlarinda diploid (2n=14) ile tetraploid (2n=28) arasinda degistigi belirlenmistir. Bununla birlikte bazi populasyonlarin ise saf olmayip farkli ploidy duzeyine sahip bitkilerden olustugu saptanmistir. Calismada incelenen tum Agropyron populasyonlarinin ise diploid (2n=14) oldugu belirlenmistir. Calismada kullanilan tum populasyonlar cekirdek DNA analiz sonuclarininda yardimiyla taksonomik olarak teshis edilmis ve isimlendirilmistir.