Molecular techniques in plant breeding, Cantho september 2006

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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

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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

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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 2006

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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 2006

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Chapter 1: Genes and genomes

DNA structureGenomes Protein structureGene structure and expression

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Cells and DNA

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Chromosomes and DNA

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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

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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

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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

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5’ end: P

3’ end: OH

This polarity is extremely important!!!!

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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

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5’ end:

5’ end:

3’ end:

3’ end:

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5’

3’

DNA Sequence: convention 5’ to 3’end, one strand (because other strand is complementary and therefore known also)

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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

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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

Organism Genomesize

Genenumber

E. coli 4.6 Mb 2800

Yeast 12 Mb 6000

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Genes and genomes

Organism Genome size

Gene number

C. elegans 100 Mb 20,000

A. thaliana 120 Mb 26,000

H. sapiens 3,000 Mb 40,000?

Z. mays 2,500 Mb 26,000?

Salamander 90,000 Mb 30,000?

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Human

Yeast

Z. mays

E. coli

Genomes 2 T.A. Brown, Bios

Genes and genomesEukaryotic genomes contain much DNA outside the genes

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

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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

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Chloroplast (cp) and

mitochondrial (mt) DNA:

Usually maternal

nuclear DNA:

From both parents (biparental)

Plant cell

Genomes

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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)

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Mitochondrial genomes

• Human: 16 kb• Yeast: 84 kb• Corn: 570 kb• Genes for:

rRNA, tRNA

proteins for ATP synthesis

human

yeast

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Chloroplast genomes

• 120 – 200 kb• Genes voor:

rRNA and tRNARNA polymeraseca. 120 proteins for photosynthesis

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Maternal inheritance of mitochondrial DNA

Maternal inheritance through egg cytoplasm, no mixing, no recombination: excellent for lineage analysis

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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.