Tomáš Nečas

Use of Combined MSAP and NGS Techniques to Identify Differentially Methylated Regions in Somaclones: A Case Study of Two Stable Somatic Wheat Mutants.

The appearance of somaclonal variability induced by in vitro cultivation is relatively frequent and can, in some cases, provide a valuable source of new genetic variation for crop improvement. The cause of this phenomenon remains unknown; however, there are a number of reports suggesting that epigenetics, including DNA methylations, are an important factor. In addition to the non-heritable DNA methylation changes caused by transient and reversible stress-responsive gene regulation, recent evidence supports the existence of mitotically and meiotically inherited changes. The induction of phenotypes via stable DNA methylation changes has occasionally great economical value; however, very little is known about the genetic or molecular basis of these phenotypes. We used a novel approach consisting of a standard MSAP analysis followed by deep amplicon sequencing to better understand this phenomenon. Our models included two wheat genotypes, and their somaclones induced using in vitro cultivation with a changed heritable phenotype (shortened stem height and silenced high molecular weight glutenin). Using this novel procedure, we obtained information on the dissimilarity of DNA methylation landscapes between the standard cultivar and its respective somaclones, and we extracted the sequences and genome regions that were differentially methylated between subjects. Transposable elements were identified as the most likely factor for producing changes in somaclone properties. In summary, the novel approach of combining MSAP and NGS is relatively easy and widely applicable, which is a rather unique feature compared with the currently available techniques in the epigenetics field.

Identification of 17 ‘Candidatus Phytoplasma pyri’ genotypes based on the diversity of the imp gene sequence

M. Bohunick a*, L. Valentov a, J. Such a, T. Ne cas, A. Eichmeier, T. Kiss and R. Cmejla Research and Breeding Institute of Pomology Holovousy Ltd., Holovousy 129, 508 01 Ho r ice; Department of Biology, Faculty of Science, University of Hradec Kr alov e, Rokitansk eho 62, 500 03 Hradec Kr alov e; Department of Pomology, Faculty of Horticulture, Mendel University in Brno, Valtick a 337, 691 44 Lednice; and Mendeleum – Institute of Genetics, Mendel University in Brno, Valtick a 334, 691 44 Lednice, Czech Republic

High-Throughput Sequencing Analysis of the Bacterial Community in Stone Fruit Phloem Tissues Infected by “Candidatus Phytoplasma prunorum”

Abstract“Candidatus Phytoplasma prunorum” (CPp) is a highly destructive phytopathogenic agent in many stone fruit-growing regions in Europe and the surrounding countries. In this work, we focused on documenting entire bacterial community in the phloem tissues of 60 stone fruit trees. Nested PCR and two real-time PCR assays were used to select CPp-positive (group A) and CPp-negative samples (group B). Afterwards, high-throughput amplicon sequencing was performed to assess bacterial community compositions in phloem tissues. The bacterial composition in phloem tissue consisted of 118 distinct genera, represented mainly by Pseudomonas, Acinetobacter, Methylobacterium, Sphingomonas, and Rhizobium. Statistics showed that CPp influenced the bacterial composition of infected plants (group A) and that the bacterial community depended on the geographical origin of the sample. This is the first work focusing on an analysis of the influence of CPp on the bacteria coexisting in the phloem tissues of stone fruit trees.