A molecular marker set combining a retrotransposon insertion and SSR polymorphisms is useful for assessing diversity in Vitis

Molecular markers, based on DNA polymorphisms, are useful tools for identifying individuals, establishing phylogenetic relationships, managing collections of genetic material or assisting breeding. In the present study, we developed a marker set to differentiate Vitis species, grapevine varieties or clones belonging to the same variety. This novel marker set combines, in four PCR amplifications, the presence/absence of a remarkable retrotransposon, Tvv1-Δ3460, inserted at its single locus and the SSR polymorphism present within its two LTRs. By studying a collection of Vitaceaeaccessions, we showed the prevalence of two allelic forms of Tvv1-Δ3460 - one of which was partially truncated - in Vitis species. Out of the twenty-five studied Vitis species, the insertion of a Tvv1-Δ3460 element was detected in twenty, including Vitis vinifera. The homozygous vs heterozygous state of the element insertion was determined by amplifying the empty site. Additionally, each Tvv1-Δ3460 LTRs included a microsatellite sequence useful for designing markers based on LTR length. The LTR-SSR markers distinguished most of the fifty-two cultivars and revealed polymorphism within five of the seven varieties studied.

CYTOLOGICAL VARIATION OF (AAG)7 REPEAT ON LETTUCE CHROMOSOMES BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

Cytological studies using fluorescence in situ hybridization (FISH) technique provides phylogenetical information in closely related taxa and have been widely applied for karyotyping and studying chromosomal organization and evolution in plant species. In the present study, FISH using a microsatellite sequence of (AAG)7 as the probe was performed in order to discriminate the chromosomes in four Lactuca species, i.e., L. sativa, L. serriola, L. saligna, and L. virosa. The experiment was carried out in April to September 2018 at Laboratory of Genetic and Plant Breeding of Breeding of Graduate School of Horticulture, Chiba University, Japan. Different distribution patterns of (AAG)n signals were shown on the chromosomes in the four Lactuca species studied, In L. sativa and L. serriola, FISH with (AAG)7 sequences revealed dispersed distribution patterns with one pair of bright signals, respectively. While in L. saligna and L. virosa, distinct signals with different intensities were observed in two pairs of chromosomes of L. saligna and five pairs of chromosomes of L. virosa. In conclusion, the AAG repeat signals could be used as cytogenetic landmarks for chromosome identification in Lactuca species.

Genetic variation of the nuclear sequences of mitochondrial origin associated with retrotransposon Tv1 insertions in Drosophila species of the virilis group

Mitochondrial DNA sequences integrated into chromosomes are a promising object for designing genetic markers for studies of phylogenesis and genomic instability. Mitochondrial genomes of D. virilis and other Drosophila species of the virilis group contain (AT)nmicrosatellites in the spacer region between the atp6 and cox3 genes, and this microsatellite sequence is one of the hallmarks of the virilis group. The nuclear genome of D. virilis contains many extended fragments of mitochondrial DNA, which in total are several times longer than the mitochondrial genome. These nuclear sequences of mitochondrial origin contain all types of mitochondrial sequences, including mitochondrial genes and the aforementioned microsatellite sequence. The presence of the (AT)nmicrosatellite allows insertion of retrotransposon Tv1, which can transpose into the (AT)n microsatellite in a site-specific manner. The Tv1 insertion into (AT)n, close to the atp6 or cox3 pseudogenes produces a unique sequence. This sequence is formed by retrotransposon Tv1 and pseudogenes atp6 or cox3. This unique sequence can be detected in the genome by a PCR-based method. We applied this method to the detection and analysis of the nucleotide variability of the pseudogenes atp6 and cox3 associated with Tv1 insertions in a D. virilis cell culture and in the genomes of four Drosophila species of the virilis group: D. virilis, D. montana, D. borealis, and D. lacicola. We discovered new events of mitochondrial sequence transfer to the nucleus in the transplanted cell culture of D. virilis, and new Tv1 insertions, having emerged during the passage of this cell line were detected in the genome of the D. virilis transplanted cell culture. We found atp6 and cox3 pseudogenes associated with insertions of retrotransposon Tv1 in the nuclear genomes of four Drosophila species from the virilis group. These chimeric sequences proved to be species-specific. The age of the Tv1 insertion into the atp6 and cox3 pseudogenes is estimated at 1.50 Ma for D. virilis, 1.31 Ma for D. lacicola, and 1.56 Ma for D. borealis. A specific situation was revealed for D. montana, in which Tv1 insertions with nearly identical 5' and 3' long terminal repeats (LTRs) were present in accessions of flies from Europe and Asia. The age of this insertion was about 300 thousand years, and the insertion was absent from the D. montana fly line from North America.

Random genomic scans at microsatellite loci for genetic diversity estimation in cold-adaptedLepidium latifolium

Lepidium latifoliumL. (Brassicaceae) grows successfully in a high-altitude cold arid environment. Little molecular data are available for this plant despite its immense ecological importance as a cold- and drought-adapted species. We used a novel approach to identify microsatellite regions using genome walker libraries, called as Random Scans at Microsatellite Regions (RaSMiR), and implemented them on genotypes collected from relatively different topographical conditions within a small geographical area. The success rate of finding a microsatellite sequence using this methodology was 100%, and on developing the RaSMiR technique itself as a molecular marker, 230 electrophoretic bands were obtained using 13 different RaSMiR primers in combination with a microsatellite sequence primer. On an average, 17 bands were obtained for each primer. The electrophoretic profiles generated by RaSMiR markers were distinct from those produced by inter-simple sequence repeat markers. This information has been documented as a dominant marker data, and has been used to construct a neighbour-joining tree that successfully distinguished all genotypes. RaSMiR is an attractive approach for the development of unique and informative microsatellites, or for genome scanning directly as a molecular marker that can potentially be employed for the estimation of genetic diversity or to identify polymorphic loci involved in adaptations particularly in the non-model species, for which sufficient genomic data are not available.

Differential Association of Plasmodium falciparum Na+/H+Exchanger Polymorphism and Quinine Responses in Field- and Culture-Adapted Isolates of Plasmodium falciparum

ABSTRACTPlasmodium falciparumisolates with decreased susceptibility to quinine are increasingly being found in malaria patients. Mechanisms involved in this resistance are not yet understood. Several studies claim that alongside mutations in the Pfcrtand Pfmdr1genes, the Pfnhe-1Na+/H+exchanger polymorphism plays a role in decreasing susceptibility. However, conflicting results on the link between the Pfnhe-1gene and quinine resistance arise from field- and culture-adapted isolates. We tested the association between Pfnhe-1, Pfcrt, and Pfmdr1polymorphisms in field- and culture-adapted isolates from various countries with theirin vitrosusceptibility to quinine. Field isolates presented a higher diversity of the Pfnhe-1microsatellite sequence than culture-adapted isolates. In culture-adapted isolates but not in field isolates, mutations in the Pfcrtand Pfmdr1genes, as well as a higher number of DNNND repeats in the Pfnhe-1gene, were associated with a higher 50% inhibitory concentration (IC50) of quinine. Furthermore, most of the culture-adapted isolates with more than one DNNND repeat in the Pfnhe-1gene also harbored mutated Pfcrtand Pfmdr1genes with an apparent cumulative effect on quinine susceptibility. This study supports the involvement of the Pfnhe-1gene in the modulation of thein vitroquinine response when associated with mutated Pfcrtand Pfmdr1genes. Culture adaptation could be responsible for selection of specific haplotypes of these three genes. Methods used for drug testing might thus influence the association between Pfnhe-1polymorphism and quinine susceptibility. However, we do not exclude the possibility that in particular settings, Pfnhe-1polymorphism can be used as a molecular marker for surveillance of quinine resistance.