Advancing Research in Agricultural and Food Sciences

Through the Use of DNA-Based Technologies

18.04.2016

AREA Conference

Dr Dimitrije Krstic

• Brief reflection on the history of molecular biology and development of DNA-based methods

• Reference to molecular biology technologies being applied in agricultural and food sciences

• Discussion of future challenges and applications of molecular tools to agricultural and food sciences

• Reference to DNA-related training activities of AREA research groups

Overview

Brief History of Molecular Biology

1869 Johann Friedrich Miescherdiscovered DNA and named it nuclein

Johann Miescher

1881 Edward Zacharias showed chromosomes are composed of nuclein.

1899 Richard Altmann renamed nuclein to nucleic acid.

Thomas Morgan

1911 Thomas Hunt Morgan discovers genes on chromosomes are the discrete units of heredity

Brief History of Molecular Biology

George Beadle

Edward Tatum

1941 – George Beadle and Edward Tatum identify that genes make proteins

Edwin Chargaff

1950 – Edwin Chargaff find Cytosine complements Guanine and Adenine complements Thymine (C:G, A:T)

1952-1953 James D. Watson and Francis H. C. Crick deduced the double helical structure of DNA

Central Dogma of Molecular Biology

Crick’s comment on the word dogma:“As it turned out, the use of the word dogma caused almost more trouble than it was worth.”

Structure

Expression

Localization

Polymerase Chain Reaction (PCR)

1983 Kary Mullis developed a PCR technique

In recognition of his improvement of thepolymerase chain reaction (PCR) techniqueHe was awarded by a Nobel Prize in 1993.

Baby Blue

1971 Kjell Kleppe and co-workersin the laboratory of H. Gobind Khoranadescribed the method in Journal of Molecular Biology

Kary Mullis Lessons for Life

In his Nobel Prize lecture, he remarked that the success didn'tmake up for his girlfriend breakingup with him shortly before.

Married and remarried 4 times.

Kary Mullis

1985 Reported being visitedby Martians

PCR technique: from 1983 to 2016• Allele-specific PCR:

• Assembly PCR

• Asymmetric PCR:

• Dial-out PCR:

• Digital PCR (dPCR)

• Helicase-dependent amplification:

• Hot start PCR:

• In silico PCR

• Intersequence-specific PCR (ISSR)

• Inverse PCR:

• Ligation-mediated PCR:

• Methylation-specific PCR (MSP):

• Miniprimer PCR

• Miniprimer PCR:

• Multiplex ligation-dependent probe amplification (MLPA)

• Multiplex-PCR:

• Nanoparticle-Assisted PCR (nanoPCR):

• Nested PCR:

• Overlap-extension PCR or Splicing by overlap extension (SOEing)

• PAN-AC

• quantitative PCR (qPCR):

• Reverse Transcription PCR (RT-PCR)

• Solid Phase PCR:

• Suicide PCR:

• Thermal asymmetric interlaced PCR (TAIL-PCR)

• Touchdown PCR

• Universal Fast Walking:

PCR application in Agriculture and Food Sciences

Product development:

• Gene discovery and cloning

• Vector construction

• Identification of transformants

• Screening and characterization

• Seed quality control

PCR application in Agriculture and Food Sciences

GMO testing:

• PCR testing for unapproved events – qualitative PCR

• PCR testing for GM content – quantitative PCR

• PCR testing for a presence of high-value commoditye.g. soybean with altered oil profile

Cultivar Identification:

• It became possible to differentiate 60 Japanese dominant rice cultivars from each other using DNA extracted and purified from rice grains.

PCR application in Agriculture and Food Sciences

Gene expression changes as a result of:

• Environmental and growth conditions

• Food and/or treatment

• Breeding schemes to improvespecific characteristics

• Pests

Quantification of the pathogen concentration in a sample

PCR application in Agriculture and Food Sciences

Hybridization Techniques

Southern blot - DNADeveloped in 1975 by Sir Edwin Southern

Northern blot - RNA

Dot-Blot Hybridization (DNA)

ELISADOT BLOT

AREA

Fluorescent In-Situ Hybridization (FISH)

DNA Chip (Microarrays)

Hybridization Techniques

Sequencing Techniques and Genomics

1977, Walter Gilbert

1977, Frederick Sanger

2.7 billion dollars up to 2003

10 million dollars per genome

Cover page, Nature

~5,000 dollars per genome

Genomics and Bioinformatics

• Phylogenetic analysis

• Information on natural diversity

• Understanding of evolutionary forces shaping species natural population structure

• Molecular epidemiology: etiology, distribution and prevention of disease

• Identification and (re)classification of pathogenmicroorganisms

• Dr Smilja Teodorovic (recruited through AREA project) - valuable help with bioinformatics

DNA and RNA extraction, q(RT)-PCR, gene expression, sequencing

FOODBIOCHEM, Pavlicevic MilicaUniversity of Parma, Department of Life Science, Italy

FOODTECH , Nemanja MirkovićUniversity of Parma, Department of Life Science, Italy

FRUITBREED, Mirjana DjokicSchool of Agriculture, Policy and Development, University of Reading, UK

WEEDSCI , Markola SaulicSchool of Agriculture, Policy and Development, University of Reading, UK

PLANTPHYS, Mrs. Ivana PetrovicInstitute of Agronomic Research (INRA), Avignon, France

AQUACARP, dr Zorka DulicNofima Research Institute, As, Norway

AREA training in DNA-based technologies

PCR-based marker methodsto establish phylogenetic relations, population variability and patterns of variability

BIODIV, dr Ivan ŠoštarićSchool of Biological Sciences, University of Reading, UK

FRUITBREED, dr Zorica Rankovic-VasicSchool of Agriculture, University of Reading, UK

AREA training in DNA-based technologies

qPCR, molecular cloning, FISH

MICROBECOL, dr Igor KljujevHelmholtz Zentrum Munchen, AMP Research Unit Microbe-Plant Interaction, Germany

Dot-blot and Northern-blot, molecular cloning, qRT-PCR

MICRODIAG, dr Ivana Stanković and dr Ana Vučurović,Department of Biology and Plant Pathology, Faculty of Sciences and Biotechnology, University of Bari, Italy

AREA training

• Collaboration with and training of regional partners

Regional collaborations:- Institute Sinisa Stankovic, Belgrade- University of Zagreb and Osjek, Croatia- University of Banja Luka, BIH

• Strengthening collaborations between EU host groups and AREA research groups

• Collaborations between AREA groups:AREA beer & chips

Future scientific challenges for AREA groups

• Training at the next level, continuous development of expertize

• Designing research for publication in journals with higher impact factor

• Increasing number of citations in future, and hence visibility of Faculty of Agriculture

• Establishing a strong scientific network through collaborations, joint grant submission (e.g. H2020), student exchange.