We developed 74 microsatellite marker primer pairs yielding 76 polymorphic loci, specific for the short arm of rye chromosome 1R (1RS) in wheat background. Four libraries enriched for microsatellite motifs AG, AAG, AC and AAC were constructed from DNA of flow-sorted 1RS chromosomes and 1,290 clones were sequenced. Additionally, 2,778 BAC-end-sequences from a 1RS specific BAC library were used for microsatellite screening and marker development. From 724 designed primer pairs, 119 produced 1RS specific bands and 74 of them showed polymorphism in a set of ten rye genotypes. We show that this high attrition rate was due to the highly repetitive nature of the rye genome consisting of a large number of transposable elements. We mapped the 76 polymorphic loci physically into three regions (bins) on 1RS; 29, 30 and 17 loci were assigned to the distal, intercalary and proximal regions of the 1RS arm, respectively. The average polymorphism information content increases with distance from the centromere, which could be due to an increased recombination rate along the chromosome arm toward's the telomere. Additionally, we demonstrate, using the data of the whole rice genome, that the intra-genomic length variation of microsatellites correlates (r = 0.87) with microsatellite polymorphism. Based on these results we suggest that an analysis of the microsatellite length variation is conducted for each species prior to microsatellite development, provided that sufficient sequence information is available. This will allow to selectively design microsatellite markers for motifs likely to yield a high level of polymorphism.
Cytoplasmically inherited characters such as resistance to viral and fungal diseases, determination of starch types, crop yield, resistance to low or high temperature often contribute to the advantageous phenotypic traits of plants. In the present study, our goal was to elucidate the genealogy of cytoplasmic genomes chloroplast and mitochondria in banana. Banana breeding is rather complicated because of the low fertility and mostly unknown origin of the edible cultivars, therefore, knowledge on the putative fertile ancestors of cytoplasmic genomes chloroplast and mitochondria would be beneficial for breeding programmes. Based on the established marker systems distinct species specific gene-pools could be identified for both chloroplast and mitochondrial genomes for Musa acuminata and Musa balbisiana wild types, respectively. Detailed analysis of the species specific chloroplast and mitochondrial gene-pools of M. acuminata and M. balbisiana revealed six chloroplast and seven mitochondrial gene-pools in the analysed accessions. Comparative analysis of the haplotypes revealed the presence of Primary Centers of origin for both chloroplast and mitochondrial genomes of both species supporting the idea of common origin of these genomes. Cytotypes representing combinations of M. acuminata chloroplast and mitochondrial gene-pools were identified in majority of the analysed hybrid cultivars. A single M. acuminata cytotype was present in the majority of the analysed cultivars, which combination was not detected in any of the wild types. On the other part a single balbisiana cytotype was identified participating in the formation of interspecies hybrids. The strong preference for the presence of certain cytoplasmic gene-pools in cultivars may indicate hundreds of years of natural as well as of farmers' selection supplementing the phenotypic traits provided by the nuclear genome. Based on the present results the present day subspecies classification of M. acuminata is also discussed.
Quercus petraea (Matt.) Liebl. and Q. robur L. hybridize frequently and occupy similar, though distinct, ecological niches. So far, genetic discrimination between these species at the molecular level has been based mainly on neutral markers. Because such markers often exhibit low species differentiation because of high genetic compatibility and exchange between Q. robur and Q. petraea at these loci, we used adaptation-related expressed genes as markers. Accordingly, we identified osmotic-stress-induced genes in a Q. petraea cell line grown under moderate osmotic stress conditions. Two subtraction libraries were established from callus cells cultured under hyperosmotic stress for 1 or 48 h. Thirty-three differentially expressed sequence tags (ESTs) (from 70 originally isolated) were classified according to their putative functions. At least five of these gene products may contribute to osmotic-stress tolerance in oak: betaine aldehyde dehydrogenase, two trans-acting transcription factors (one abscsic acid (ABA)-responsive, the other ABA-independent), a glutathione-S- transferase and a heat-shock cognate protein. Seven genes were selected based on their putative function and their expression monitored in vivo. Leaf tissue from Q. petraea and Q. robur plantlets grown hydroponically under hyperosmotic conditions was harvested after 0, 1, 6, 24 or 72 h and analyzed by real-time polymerase chain reaction (PCR). We found indications of osmotic stress adaptation in Q. petraea based on up-regulation of genes related to protective functions, whereas down-regulation of these genes was evident in Q. robur. Thus, genetic markers related to adaptive traits may be useful for differentiating Q. petraea and Q. robur genotypes.
Oilseed rape is considered relatively recalcitrant for genetic modification. This work was performed to establish conditions for effective transformation and regeneration of commercially used cultivars Campino, Haydn, Heros, Hunter and Topas (Brassica napus L.). Cotyledonary petioles and hypocotyls (obtained from the seedlings grown under dark conditions) as a source of explants were used. Our experiments revealed that a lower selection pressure in combination with postponing of selection by 14 days after co-cultivation resulted in recovery of transgenic plants from all cultivars. Transformants were obtained with efficiency from 1 to 5.5%. Histochemically GUS-positive plants were analysed by PCR using primers corresponding to the internal fragments of gus and nptII genes. The transgenic nature was confirmed by Southern blot analyses using a specific nptII probe. This work enriches the list of oilseed rape cultivars available for genetic modification.
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