Amplified fragment length polymorphism (AFLP) analysis of 24 in vitro regenerated rye plants was performed in order to evaluate the somaclonal variation rate in this species and to identify rye genomic regions where mutations are preferentially promoted by in vitro culture processes. Regenerated plants were obtained from cell lines derived from immature embryos and plants were regenerated by somatic embryogenesis. Twenty-three regenerants showed variation when compared against sibling plants obtained from the same cell line. A total number of 887 AFLP markers were scored, and 8.8% identified the same polymorphism in plants obtained independently from different cell lines, revealing putative mutational hot spots. Using controlled crossings and analysis of the corresponding progenies, we were able to verify the genetic stability in the next generation for only five of these polymorphisms. The nucleotide sequence of the AFLP amplicon of four of the polymorphic markers was obtained, but only the sequence of two markers was clearly identified in the databases. The sequence of marker A1-303 was identified as part of a tandemly repeated sequence, the 120-bp family, which is located at telomeric regions and is widely distributed among rye chromosomes. The marker A5-375 showed high similarity with regions of Angela retrotransposons.

A previous Lens genetic map was improved by adding 31 molecular genetic markers, reaching a total of 190 markers with undistorted segregation. Data were obtained from the segregational analysis of 113 F2 plants generated from a single hybrid of Lens culinaris ssp. culinaris × L. c. ssp. orientalis. The added markers are predominantly codominant (15 SSRs, five CAPSs, four presence-absence polymorphisms, three length polymorphisms, two RAPDs, and two SRAPs). At a LOD score of 3.0, the 190 markers were grouped into eight linkage groups (LG) covering 2,234.4 cM, with an average distance between markers of 12.28 cM. This linkage map has reduced the numbers of linkage groups from ten in the previous map to eight. Most of the added markers must be functional markers since primers were mostly designed to amplify transcribed sequences. Some of the amplicons were sequenced to test if they were functional markers. One of the sequences showed homology with the Pisum TFL1a gene, involved in the transition from vegetative to flowering stages. This lentil gene was located in the LG 1 thanks to the presence of a polymorphic microsatellite in the first intron of the gene. Since L. culinaris ssp. orientalis is the primary source of additional genetic variability for lentil, this improved map could help in the use of such variability in lentil breeding programs.

Objective: Peripheral blood is the most promising source of RNA biomarkers for diagnostic and epidemiological studies, because the presence of disease and prognostic information is reflected in the gene expression pattern. Quality RNA is used by a number of different downstream applications, so the selection of the most appropriate RNA stabilization and purification method is important. We have analyzed the RNA purified from 300 blood samples from 25 donors processed by two technicians using three methodologies with Tempus and PaxGene tubes. Results: The best quality sample results were obtained with the Tempus Spin RNA Isolation Kit and the PaxGene Blood miRNA Kit, although larger amounts of RNA were obtained with the Tempus Spin RNA Isolation Kit. Lower Cq values were observed for RNA and miRNA genes in samples that were tested with PaxGene Blood miRNA Kit and Tempus Spin RNA Isolation Kit respectively. We identify the Tempus Spin RNA Isolation Kit as the most robust methodology, whilst the MagMax for Stabilized Blood Tubes RNA Isolation Kit showed the most instability. For biobanks, which process a large cohort and conduct epidemiological studies, the Tempus Spin RNA Isolation Kit is the most appropriate methodology. The study demonstrates the robustness of real-life procedures.

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