Molecular techniques in plant breeding, Cantho september DAY 1: Molecular biology, the basics Chapter 1: DNA structure and gene expression, nuclear and organel genomes 53 Chapter 2: DNA replication, PCR, electrophoresis, DNA sequence analysis 33 Chapter 3: Organel and ITS sequence, DNA databases, phylogenetic analysis 46 Pract.: DNA BLAST, alignment, phylogeny Contents

Molecular techniques in plant breeding, Cantho september DAY 2: Methods for the analysis of DNA polymorphisms Chapter 4: Recombinant DNA, restriction enzymes, cloning, transformation 21 Chapter 5: Hybridisation, RFLP, micro-array & SNP analysis 33 Chapter 6: PCR methods for analysis of DNA polymorphisms 29 Contents

Molecular techniques in plant breeding, Cantho september DAY 4: Marker assisted breeding Feedback on molecular techniques Chapter 7: The use of molecular markers in breeding 18 Chapter 8: Fingerprints, diversity analysis, specific markers 19 Chapter 9: Genetic linkage, genetic maps, Introgression, QTL analysis 42 Pract.: Demonstration AFLP, SSR, QTL analysis, genetic maps Contents

Molecular techniques in plant breeding, Cantho september DAY 5: Functional genomics, transformation technology Chapter 10: Plant transformation technology 29 Chapter 11: Functional genomics 11 Chapter 12: Transgenic crop plants 31 Examination Contents

Molecular techniques in plant breeding, Cantho september Chapter 1: Genes and genomes DNA structure Genomes Protein structure Gene structure and expression

Molecular techniques in plant breeding, Cantho september Cells and DNA

Molecular techniques in plant breeding, Cantho september Chromosomes and DNA

Molecular techniques in plant breeding, Cantho september DNA: Chemical Composition Two types of nitrogen-containing bases comprise the chemical structure of DNA: - purines = adenine and guanine, A & G - pyrimidines = thymine and cytosine, T & C

Molecular techniques in plant breeding, Cantho september DNA: Chemical Composition Nucleotides = building blocks of DNA = phosphate + sugar + base Nucleoside = sugar +base Sugar = 5 carbon deoxyribose Phosphodiester bonds link sugar molecules to phosphate groups

Molecular techniques in plant breeding, Cantho september

Molecular techniques in plant breeding, Cantho september DNA: Chemical Structure DNA = polynucleotide = a chain of bases Orientation of sugar-phosphate linkages = 5’ to 3’ as the phosphate attached to the 5’ carbon of one sugar is linked to the 3’ carbon of the next sugar Purine and pyrimidine bases are linked to the 1’ carbon of sugar

Molecular techniques in plant breeding, Cantho september ’ end: P 3’ end: OH This polarity is extremely important!!!!

Molecular techniques in plant breeding, Cantho september DNA: Chemical Structure DNA consists of two polynucleotide chains which run 5’ to 3’ in opposite directions = antiparallel DNA chains are held together by hydrogen bonds between bases DNA bases pair by Chargaff’s rules: - Adenine (A) pairs with Thymine (T) by 2 H-bounds - Guanine (G) pairs with Cytosine (C) by 3 bounds

Molecular techniques in plant breeding, Cantho september ’ end: 3’ end:

Molecular techniques in plant breeding, Cantho september ’ 3’ DNA Sequence: convention 5’ to 3’end, one strand (because other strand is complementary and therefore known also)

Molecular techniques in plant breeding, Cantho september Genome Size Complex organisms have large genomes = genetic contents of a cell Genomic size increases with evolutionary complexity (in general!) Size of DNA is measured in kb = kilobase pairs Size of large genomes is measured in Mb = megabase pairs

Molecular techniques in plant breeding, Cantho september Genes and genomes Gene = unique sequence of DNA bases, coding for a protein Alleles = different variants of a gene in different organisms or on the homologous chromosomes

Molecular techniques in plant breeding, Cantho september Genes and genomes

Molecular techniques in plant breeding, Cantho september Human Yeast Z. mays E. coli Genomes 2 T.A. Brown, Bios Genes and genomes Eukaryotic genomes contain much DNA outside the genes

20 Chromosomes of a human being: 2 copies of each chromosome, ordered by size on a slide

Molecular techniques in plant breeding, Cantho september Chromosomes and DNA Eukaryotes are diploid (2 homologous chromosomes, with alleles of genes) Eukaryotes have linear chromosomes in their nucleus This picture shows a duplicated chromosome= 2C = 2 identical chromatids, the scheme unravels one of those

Molecular techniques in plant breeding, Cantho september Chloroplast (cp) and mitochondrial (mt) DNA: Usually maternal nuclear DNA: From both parents (biparental) Plant cell Genomes

Molecular techniques in plant breeding, Cantho september Genomes Prokaryotes have a circular genome and often also small circles = plasmids Mitochondria (and chloroplasts) in eukaryotic cells have their own genome = circular (endosymbiont hypothesis) Prokaryote was swallowed by an early eukaryote and degenerated, it now provides energy (mitochondria) or performs photosynthesis (chloroplast)

Molecular techniques in plant breeding, Cantho september Mitochondrial genomes Human: 16 kb Yeast: 84 kb Corn: 570 kb Genes for: rRNA, tRNA proteins for ATP synthesis human yeast

Molecular techniques in plant breeding, Cantho september Chloroplast genomes 120 – 200 kb Genes voor: rRNA and tRNA RNA polymerase ca. 120 proteins for photosynthesis

Molecular techniques in plant breeding, Cantho september Maternal inheritance of mitochondrial DNA Maternal inheritance through egg cytoplasm, no mixing, no recombination: excellent for lineage analysis

Molecular techniques in plant breeding, Cantho september Organel DNA for tracing back Mystery of Russian tsar-family: bones in grave could be identified based on comparison of mitochondrial DNA with that of living descendants (through the maternal line). Many copies (many circles per organel, many organels per cell) of the DNA allow efficient analysis.