Introduction

The Genetic laboratory was established in 1998 at the Agricultural Institute of Slovenia under the leadership of assoc. prof. dr. Vladimir Meglič  at the Crop Science Department. Today, the genetic laboratory is part of the Crop Science Department (OPVGŽ). Within the genetic laboratory, we have laboratory for tissue culture and the laboratory for plant physiology.

The genetic laboratory are using researchers from the OPVGŽ, Plant Protection Department (OVR) and the Animal Production Department (OŽ). The head of the laboratory is dr. Barbara Pipan (active link to , who is as well responsible for the OPVGŽ laboratory work. She is assisted by dr. Barbara Gerič Stare, who coordinates the work of OVR and mag. Andreja Opara, who is responsible for the OŽ research work.

The genetic laboratory facilities enable us to perform wide arrays of analyses in  the field of molecular biology, genetics, population genetics of plants and animals, environmental genetics, diagnostics and to conduct general genetic analyses using different types of marker systems (biochemical, molecular). The major research equipment in our laboratory is:
• tissueLyser, (Qiagen),
• automated system for the extraction of nucleic acids (KingFisher and MagMax, Thermo Fisher Scientific),
• QIAxcel Advanced capillary electrophoresis (Qiagen),
• analyzer of nucleic acids and proteins (Agilent),
• various types of cyclic thermostats (ABI Veriti, SureCycler Agilent, ABI9700), including RT-PCR (ABI7500 with fast and HRM upgrade),
• fluorometer (Qubit 3.0),
• heat shaker (for macroarray, GeneTop Biotech),
• horizontal electrophoresis with a gel imaging system (Biometra, GeneGenius),
• additional equipment available on CFGBC/MF
 (Nanodrop) and BF (Genetic analyzer ABI3130XL, 2D-gel electrophoresis) for sharing with Agricultural institute of Slovenia as a member of the consortium,

• and within the laboratory for ecophysiology we have portable photosynthesis system (LI-COR 6400XT), leaf area meter ((LI3100C) and leaf canopy meter (LAI220C).

 

Scientific and expert publications related to genetic laboratory research results are available at the Agrobiodiversity research program site. 

OPVGŽ (Crop Science Department) research conducted in the genetic laboratory

Genetic structure and diversity of agricultural species

Genetic research is focused on studying genetic diversity resources of agronomically important crops including: cereals (Avena sp., Triticum sp., Triticosecale, Hordeum sp.), pseudocereals (Fagopyrum sp.) and brassicas (Brassica napus, B. rapa, Sinapis arvensis). In addition, our work includes grapevine, blueberries, apple trees, ambrosia, dandelions, sesame, clovers, maize, sweet potatoes and runner beans. More detailed studies of genetic diversity, genetic structure and molecular variability, especially in relation to plant breeding procedure using functional DNA markers include common bean (Phaseolus vulgaris L.) and potato (Solanum tuberosum L.). Our aim is to identify genotypes with (multiple) alleles and genes that are associated with the favourable agronomically important traits. Interpretation of the results of genetic analysis using appropriate bioinformatics processing from the population genetics through parameters of genetic diversity provides useful information about genetic diversity between genotypes within/between varieties; about their genetic structure and genetic groups and their origin. For environmental genetics, those parameters could indicate to the out-crossing level, gene flow potential and in general the relationships of genotypes comparing different taxonomic groups.

Genetic structure of common bean genotypes (10 genetic groups/colours) from 12 geographic origins

For the varietal identification, verifying varietal identity and purity and identifying atypical representatives in seed and certified material, the use of molecular markers is highly applicable. Related to that, the project entitled " Establishment of a system for the use of DNA markers for genetic identification and verification of varietal authenticity and purity of major cereals and brassicas as a basis for the quality production of seeds, food and feed" has been started at the OPVGŽ, for using DNA markers as fast and effective tool for distinguishing genotypes at different levels.

Genetic relations among commercial genotypes of oilseed rapes varieties (left) and distribution of oilseed rape genotypes in factorial correspondence analysis on the basis of 45 loci among whole genome

In general, DNA marker systems represent reliable, repeatable, financially acceptable and effective methods for solving many of the issues in agriculture, which cannot be answered phenotypically / morphologically. Depending on the characteristics of the problem and the objectives of the tasks, the results of genetic analyses would be useful for farmers, commercial companies, breeders, researchers, certification services, inspection services and various ministries (Ministry of environment and spatial panning; Ministry of agriculture, forestry and food).

Genotypes of common bean (red points) with (partial) resistance to virus (es) BCMV/BCMNV (I gene)

 

Selected publications

PIPAN, Barbara, ZUPANČIČ, Maša, BLATNIK, Eva, DOLNIČAR, Peter, MEGLIČ, Vladimir. Comparison of six genomic DNA extraction methods for molecular downstream applications of apple tree (Malus X domestica). Cogent food & agriculture, ISSN 2331-1932, 2018, no. 4, str. 1-10, graf. prikazi. https://www.tandfonline.com/doi/pdf/10.1080/23311932.2018.1540094?needAccess=true, doi: 10.1080/23311932.2018.1540094

PIPAN, Barbara, ŽNIDARČIČ, Dragan, KUNSTELJ, Nataša, MEGLIČ, Vladimir. Genetic evaluation of sweetpotato accessions introduced to the central European area. Journal of agricultural science and technology, ISSN 1680-7073, 2017, vol. 19, iss. 5, str. 1139-1150. http://jast.modares.ac.ir/article_17081_cd7e33edb6c841cd3a42a45aa01e5d4f.pdf

PIPAN, Barbara, ŽNIDARČIČ, Dragan, MEGLIČ, Vladimir. Evaluation of genetic diversity of sweet potato [Ipomoea batatas (L.) Lam.] on different ploidy levels applying two capillary platforms. The journal of horticultural science & biotechnology, ISSN 1462-0316, 2017, vol. 92, iss. 2, str. 192-198, doi: 10.1080/14620316.2016.1249963

MARAS, Marko, PIPAN, Barbara, ŠUŠTAR VOZLIČ, Jelka, MEGLIČ, Vladimir, et al. Examination of genetic diversity of Common bean from the Western Balkans. Journal of the American Society for Horticultural Science, ISSN 0003-1062. [Print ed.], 2015, letn. 140, iss. 4, str. 308-316. 

PIPAN, Barbara, ŠUŠTAR VOZLIČ, Jelka, MEGLIČ, Vladimir. Genetic differentiation among sexually compatible relatives of Brassica napus L. = Genetska diferenciacija izmedu seksualno kompatibilnih srodnika kod vrste Brassica napus L. Genetika : časopis Saveza društava genetičara Jugoslavije, ISSN 0534-0012, (Acta biologica Iugoslavica), 2013, vol. 45, no. 2, str. 309-327, ilustr., doi: 10.2298/GENSR1302309P

MEGLIČ, Vladimir, PIPAN, Barbara. Spatial and temporal assessment of Brassica napus L. maintaining genetic diversity and gene flow potential: an empirical evaluation. V: EL-ESAWI, Mohamed A. (ur.). Brassica germplasm: charecterization, breeding and utilization. London: IntechOpen. 2018, str. [27]-44, ilustr. https://www.intechopen. com/books/brassica-germplasm-characterization-breeding-and-utilization/spatial-and-temporal-assessment-of-brassica-napus-l-maintaining-genet ic-diversity-and-gene-flow-pote, doi: 10.5772/intechopen.74570

Marker assisted selection in breeding of agricultural crops

The introgression of genes for favourable traits is performed through hand cross-pollination between selected elite genetic resources and further selection. Genetic mapping studies utilizing sources of resistance and other desirable traits have enabled identification and characterization genes underlying these traits, followed by development of molecular markers for tagging them. Molecular marker assisted selection enables precise characterization of breeding materials, facilitates the breeding process by speeding up the selection process and enables creation of superior genotypes by gene pyramiding.

Agricultural Institute of Slovenia has a long tradition in breeding of agricultural crops, with established long-term programs for potato, common bean, buckwheat and fodder crops. The role of genetic laboratory is to provide support for in-house breeding programs by;

  • Marker assisted selection to facilitate selection of progeny with desirable traits utilizing markers associated with desirable traits and specific resistance genes to diseases relevant to our production area
  • Marker assisted selection to facilitate gene pyramiding
  • Genome wide association studies (GWAS) and mapping of quantitative trait loci (QTL) enable identification of loci, genes and genetic markers associated with the traits of interest

 

 

Selected publications

M. Zupin. Response to drought stress in common bean (Phaseolus vulgaris L.): QTL mapping, 2017, Ph. D. thesis

P. Dolničar. Pyramiding of R genes responsible for resistance to Potato virus Y and potato late blight, 2016, Ph.D. thesis 

Varieties of potato and common bean

 

Studies of crop plant responses to biotic and abiotic stress

Crop plants are affected by biotic and abiotic stress that affects quantity and quality of the yield in the field as well as during postharvest procedures. Plant responses can be studied on a physiological level whereas molecular biology techniques enable studies down to the expression of genes, proteins and metabolites. Novel high-throughput techniques enable a system-wide examination of gene and protein expression and form a basis for studies of plant metabolic pathways utilising bioinformatics and systems biology approaches.  At the Crop Science Department we study responses of agricultural plants to stressors, relevant for our production area.

  • In the area of molecular plant pathology, we study the interaction of potatoes with damaging pathogens in our production area.  We study how storage conditions affect the necrotic symptom development in PVYNTN infected potatoes on a transcriptomic level. We study the underlying resistance responses of potato plants containing specific R genes to Phytophtora infestans and common bean plants with respect to resistance to anthracnose (Colletotrichum lindemuthianum).

 

  • In the area of plant response to abiotic stress we study common bean response and adaptation to drought and subsequent rehydration, on the level of physiology, transcriptomic and proteomic levels, as well as protease activity.

 

 

Selected publications

A. Sedlar, B. Gerič Stare, I. Mavrič Pleško, P. Dolničar, M. Maras, J. Šuštar-Vozlič, Š. Baebler, K. Gruden, V. Meglič. Expression and regulation of programmed cell death associated genes in systemic necrosis of PVYNTN susceptible potato tubers Plant Pathology DOI: 10.1111/ppa.12818 http://onlinelibrary.wiley.com/doi/10.1111/ppa.12818/full

T. Zadražnik, A. Moen, W. Egge-Jacobsen, V. Meglič, J. Šuštar-Vozlič.  Towards a better understanding of protein changes in common bean under drought: A case study of N-glycoproteins Plant Physiology and Biochemistry, 118 (2017), pp. 400-412 https://doi.org/10.1016/j.plaphy.2017.07.004

T. Zadražnik, W. Egge-Jacobsen, V. Meglič, J. Šuštar-Vozlič. Proteomic analysis of common bean stem under drought stress using in-gel stable isotope labeling J. Plant Physiol., 209 (2017), pp. 42-50 https://doi.org/10.1016/j.jplph.2016.10.015

 

M. Zupin, A. Sedlar, M. Kidrič, V. Meglič. Drought-induced expression of aquaporin genes in leaves of two common bean cultivars differing in tolerance to drought stress. J. Plant Res., 130 (2017), pp. 735-745 10.1007/s10265-017-0920-x

 

T. Zadražnik, K. Hollung, W. Egge-Jacobsen, V. Meglič, J. Šuštar-Vozlič. Differential proteomic analysis of drought stress response in leaves of common bean (Phaseolus vulgaris L.) J. Proteomics, 78 (2013), pp. 254-272 https://doi.org/10.1016/j.jprot.2012.09.021

 

Plant tissue culture

 

In our laboratory, plant tissue cultures are used:

  • as a complement to conventional breeding methods,
  • mikropropagation of virus-free plants, when a large number of plants can be grown over a relatively short period of time,
  • virus elimination of  infected plants,
  • basic biological studies,
  • saving of inheritance (gene bank: in vitro plants and microtubers); it is about feeding autochthonous varieties and all the materials we need in raising new varieties nad research.

 

OVR (Plant Protection Department) research conducted in the genetic laboratory

Genetic variability of plant pathogens and pests

 

The damage to plants caused by pests including viruses, bacteria, fungi, nematodes, insects and mites causes great yield losses and economic damage in agricultural production. Plants and pests identify and react to each other at the molecular level. With molecular biology tools genes and the gene products involved in plant-pest interaction can be identified. Even small differences in these key molecules, both in plants and in pests, can be the basis for plant resistance or virulence of pests. At Plant Protection Department we are identifying characteristics of these key molecules:

 

Nematology:

Identification of tropical root knot nematodes (Meloidogyne spp.) is challenging due to hybrid origin of these parthenogenetic species. We evaluated the suitability of different regions of DNA for molecular markers.

 

Like other pathogens, plant parasitic nematodes employ effector proteins to alter plant cellular functions and successfully infect their hosts. We have assessed molecular variability of cell wall-modifying protein (expansins and pectate lyases) in potato cyst nematodes Globodera rostochiensis and G. pallida.

 

Entomology:

With molecular typing based on mtDNA and microsatellite markers we can track the origin of the western corn rootworm Diabrotica virgifera virgigera, an economically important pest of maize in Northern America and Europe including Slovenia.

 

Bacteriology:

We explored the presence of Agrobacterium spp., a causative agent of the crown gall disease. Based on a recA allele sequence analysis isolated strains of Agrobacterium species complex were assigned to genomic species G1 and G4, and new alleles were recognized.

 

Virology:

Potato spindle tuber viroid (PSTVd) can be transmitted with ornamental plants. Sequence analysis of PSTVd revealed new variants in samples from Slovenia.

 

Selected publications

GERIČ STARE, Barbara, STRAJNAR, Polona, SUSIČ, Nik, UREK, Gregor, ŠIRCA, Saša. 2017. Reported populations of Meloidogyne ethiopica in Europe identified as Meloidogyne luci. Plant disease, vol. 101, no. 9, str. 1627-1632. https://apsjournals.apsnet.org/doi/10.1094/PDIS-02-17-0220-RE

ALI, Shawkat, MAGNE, Maxime, CHEN, Shiyan, CÔTE, Olivier, GERIČ STARE, Barbara, OBRADOVIC, Natasa, JAMSHAID, Lubna, WANG, Xiaohong, BÉLAIR, Guy, MOFFETT, Peter. 2015. Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses. PloS one, ISSN 1932-6203. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115042

ŠTRUKELJ, Melita, MODIC, Špela, SEDLAR, Aleš, WINTER, Stephan, RAZINGER, Jaka. The use of microsatellite markers as a tool for distinction between western corn rootworm (Diabrotica virgifera virgifera [Coleoptera: Chrysomelidae]) populations. V: TRDAN, Stanislav (ur.). Zbornik predavanj in referatov 13. slovenskega posvetovanja o varstvu rastlin z mednarodno udeležbo, Rimske Toplice, 7.-8. marec 2017 = Lectures and papers presented at the 13th Slovenian Conference on Plant Protection with International Participation, Rimske Toplice, March 7-8, 2017. Ljubljana: Društvo za varstvo rastlin Slovenije: = Plant Protection Society of Slovenia. 2017, str. 358-365. http://www.dvrs.bf.uni-lj.si/spvr/2017/zbornik_2017.html

LAMOVŠEK, Janja, GERIČ STARE, Barbara, UREK, Gregor. 2014. Isolation of non-pathogenic Agrobacterium spp. biovar 1 from agricultural soils in Slovenia. Phytopathologia Mediterranea, vol. 53, no. 1, str. 130-139. http://fupress.net/index.php/pm/article/view/12706

VIRŠČEK MARN, Mojca, MAVRIČ PLEŠKO, Irena, GERIČ STARE, Barbara. 2013. Variability of potato spindle tuber viroid isolates from ornamental hosts in Slovenia. Journal of plant pathology, vol. 95, no. 2, str. 411-415. http://www.sipav.org/main/jpp/index.php/jpp/article/view/2830

 

OŽ (Animal Production Department) research conducted in the genetic laboratory

Genetic analysis in cattle

 

In the genetic laboratory, we perform analyses for verification of origin, using a standard ISAG set (12 microsatellites).

 

Determining recessive genetic traits (mutations)

CVM – Complex Vertebral Malformation

BLAD - Bovine Leucocyte Adhesion Deficiency

RF - Red factor

Pedigree testing in cattle

We are verifying cattle pedigrees with genetic markers and software within the information system Cattle at the Agricultural Institute of Slovenia.

Method STR (Microsatellites)

We check the origin of cattle with microsatellites. We are involved in the ISAG international comparison test, where we are among the most reliable laboratories (certificate).

 

 

SNP method (genotyping with SNP chip)

We are accredited by ICAR for verification of origin using SNP data.

 

Selected publications

 

LOGAR, Betka, KAVAR, Tatjana, MEGLIČ, Vladimir. Detection of recessive mutations (CVM, BLAD and Red Factor) in Holstein bulls in Slovenia = Določanje recesivnih mutacij (CVM, BLAD in alel za rdečo barvo) pri bikih črno-bele pasme v Sloveniji. Journal of central european agriculture : JCEA, ISSN 1332-9049. [Online ed.], 2008, vol. 9, no. 1, str. 101-106, graf. prikazi. http://www.agr.hr/jcea/issues/jcea9-1/index.php

 

OPARA, Andreja, RAZPET, Andrej, LOGAR, Betka. Breed assignment test of Slovenian cattle breeds using microsatellites. V: PETRIČ, Nežika (ur.). Livestock production as a technological and social challenge, (Acta agriculturae slovenica, ISSN 1854-4800, Supplement, 2012, 3). Ljubljana: Biotechnical Faculty. 2012, suppl. 3, str. 167-170. http://aas.bf.uni-lj.si/zootehnika/supl/3-2012/PDF/3-2012-167-170.pdf

 

OPARA, Andreja. Monogenske napake pri črno-beli, rjavi in lisasti pasmi goveda = Monogenic errors in Holstein, Brown and Simmental cattle breeds. V: ČEH, Tatjana (ur.), KAPUN, Stanko (ur.). Zbornik predavanj = Proceedings of the 26th International Scientific Symposium on Nutrition of Farm Animals [being] Zadravec-Erjavec Days 2017, 9th and 10th November 2017. Murska Sobota: Kmetijsko gozdarska zbornica Slovenije, Kmetijsko gozdarski zavod. 2017, str. 167-174

 

OPARA, Andreja. Uporaba DNK označevalcev za preverjanje porekla pri govedu. Govedorejec : glasilo Govedorejskega društva, mar. 2010, št. 18, str. 17, ilustr. [COBISS.SI-ID 3287912] http://www.kmetijskizavod-celje.si/images/upload/2010/254_gov_2010_web_optimal.pdf

 

OPARA, Andreja, RAZPET, Andrej, LOGAR, Betka, BABNIK, Drago. Določitev pasme goveda na podlagi mikrosatelitnih označevalcev = Determining cattle breeds on the rasis of microsatellite markers. V: ČEH, Tatjana (ur.). Zbornik predavanj = Proceedings of the 21st International Scientific Symposium on Nutrition of Farm Animals : [being] Zadravec-Erjavec Days 2012, Radenci, November 8th and 9th, 2012, 21. mednarodno znanstveno posvetovanje o prehrani domačih živali, Zadravčevi-Erjavčevi dnevi 2012. Murska Sobota: Kmetijsko gozdarska zbornica Slovenije, Kmetijsko gozdarski zavod Murska Sobota. 2012, str. 115-118. https://www.govedo.si/files/janezj2/ZED_2012/dolocitev_pasme_goveda_na_podlagi_mikrosatelitnih_oznacevalcev.pdf

 

OPARA, Andreja, RAZPET, Andrej. Population studies for 6 cattle breeds and 11 microsatellite loci. V: ARCHIBALD, Alan (ur.). 32nd Conference of the International Society for Animal Genetics, 26th - 30th July 2010, Edinburgh, Scotland : Programme and Abstract Book. [Edinburgh]: [s. n.]. 2010, str. 60

 

OPARA, Andreja. Preverjanje porekla in določanje genetske pripadnosti osebkov trem glavnim pasmam goveda v Sloveniji : magistrsko delo = Pedigree testing and assignment test in three main cattle breeds in Slovenia : M. Sc. thesis. Ljubljana: [A. Opara], 2016. IX, 59 f., ilustr. http://www.digitalna-knjiznica.bf.uni-lj.si/podiplomski_studij/md_opara_andreja.pdf

 

OBŠTETER, Jana, LOGAR, Betka, OPARA, Andreja, JENKO, Janez. Parentage verification using imputed microsatellite and SNP data in Slvoenian brown swiss population. V: Proceedings of the 2016 Interbull Meeting, Puerto Varas, Chile, October 24 - 28, 2016, (INTERBULL Bulletin, ISSN 2001-340X, No. 50, 2016). [Uppsala: Interbull Centre. 2016], str. 108-110. https://journal.interbull.org/index.php/ib/article/view/1398

 

 

Molecular analyses in apiculture

 

In the Apiculture group, we explore the options for routine genotyping of honey bee queens origin and genetic determination of the entomological signatures as a tool to detect the counterfeit honey and other products that are stated to contain honey bee resources.

Selected publications

 

MOŠKRIČ, Ajda, MOLE, Katarina, DOVČ, Peter, PREŠERN, Janez. Izolacija dednine iz neinvazivnih bioloških virov pri maticah kranjske čebele (Apis mellifera carnica) = Isolation of genomic dna from non-invasive samples of carniolan honey bee queens (Apis mellifera carnica). V: SMODIŠ ŠKERL, Maja Ivana (ur.), MOŠKRIČ, Ajda (ur.). Zbornik referatov = Book of proceedings, 3. Znanstveno posvetovanje o čebelah in čebelarstvu [tudi] Poklukarjevi dnevi, Ljubljana, 4.-5. oktober 2018 = 3rd Scientific Symposium on Bees and Beekeeping [being] Poklukar's Days, in Ljubljana, 4th-5th October 2018. Ljubljana: Slovensko akademsko čebelarsko društvo: Kmetijski inštitut Slovenije. 2018, str. 85-92. http://www.kis.si/f/docs/Poklukarjevi_dnevi_2018/ZBORNIK_PRISPEVKOV_PD_2018.pdf.