1. GENETICS genetic mapping, classical approaches to study gene function

2. Basic aims: uncovering gene function understand mechanisms of morphogenesis, development, metabolism, physiology etc. in connection with coordinated gene expression breeding production of plants (organisms) with improved characteristics or their combination

3. Terminology Gene segment of genomic information that specifies a trait basic unit of heridity in living organisms Genotype + environment + ? = phenotype Interactions between genes/proteins (epistasis – metabolic and signal pathways)

4. Allele – form of a gene dominant vs. recesive genesis of new alleles by mutations Locus – location of a gene on a chromosome Genetic linkage – inheriting of certain genes (their alleles) jointly, because they reside on the same chromosome (gene distance cM = % of recombinant gametes) Genetic (likage) maps x physical maps

5. - varying likelyhood of recombination – cM (0-50 cM) What sequences are with lower recombination probability? Genetic (linkage) and physical maps differ

6. Genetic likage x crossing-over during meiosis Genetic likage x crossing-over during meiosis 1.Cytologic event Parental chromosomes Meiosis Without Crossing-over Gametes 1 2 3 4 Not recombinant Recombinant 2. Genetic result Parental Genotype (heterozygous Aa and Bb ) Locus A Locus B Meiosis Gametes Not recombinant ( same as parental genotype ) Recombinant ( new )

7. Genetic maps - genes - markers (= any detectable feature with known position on chromosoms ) (identifiable)

8. Genetics classical (direct) x reverse Direct – from a trait (phenotype) to identification of corresponding gene Reverse – from a gene to phenotype (study of gene function by mutagenesis, modulated expression, …) - both approaches need mutants

9. Mutagenesis Classical: –chemical m. – EMS (ethane metyl sulfonate; point mutations) –physical m. – RTG, gama... (usually short deletions) –wide spektrum of affects (regulation, interaction) –even dominant mutations, resamble natural mutations, difficult/expensive identification of mutated gene Direct – looking for certain phenotype in mutant population Reverse – targeted mutagenesis/modification of selected gene

10. Mutagenesis Advanced: –insertional mutagenesis – T-DNA, transposons –random insertions –allows simple determination of the site of insertion = mutation attached to a tag (inserted sequence) –various stratagies for gene isolation

11. Gene isolation based on phenotypic change caused by insertion Insertional inactivation - T-DNA tagging - transposon tagging Activation mutagenesis inserted sequence contain promoter or enhancer that can activate expression of adjacent otherwise inactive gene Promotor, enhancer-trap - T-DNA with reporter gene without promoter (with minimal promoter) original gene selection based on reporter gene expression

12. Based on genetic map and segregation analysis mapping – determination of position of the mutation in genetic map by cosegregation with genetic markers (polymorphic between parental genotypes) Identification of mutated sequence – chromosom walking, sequenation, comparison with WT Identification of mutated gene

13. Point mutations, short deletions 1) Based on genetic map and segregation analysis + chromosom walking, sequencing (long, expensive) 2)Using NGS (quick, moderate expensive) - even in unknown genomes!!! - mixed samples (back crosses) - comparisons of frequencies of similar oligomers NordströmNordström et al. Nature Biotech.2013 Identification of mutated gene (responsible for the mutation)

14. Identification of mutated gene Insertional mutagenesis: sequencing of flanking region (low template concentration for direct sequencing!) TAIL PCR (Thermal Asymmetric InterLaced PCR) adaptor PCR plasmid rescue iPCR

15. TAIL PCR : 1. three PCR (optimized T a ) with specific primer SP1-3 + certain AP 2. product sequencing SP1-3: complementary to inserted DNA AP: arbitrary (degenerated) primer - several universal types, high P of anealing near insertion SP1 SP2 SP3 AP SP1AP SP2 AP SP3 AP EE Adaptor PCR : EE SP1 SP2 SP3 SAP AP 1.cleavage (restriction endon., E) 2.ligation of adaptors 3.2-3 PCR (spec. adapt. primer + spec. primers complementary to inserted DNA) 4. product sequencing

16. 1.cleavage (E) 2.circularization (ligation) 3.transformation E.coli (ori, R) 4.multiplication in bacteria 5.sequencing oribla/nptIII E E oribla/nptIII E Plasmid rescue : Inverse PCR : EE E 1.cleavage (E) 2.circularization (ligation) 3.PCR 4.sequencing plasmid E

17. Collections of insertion mutants -publicly available (Arabidopsis, rice, …) -insertions in different positions in genome – practically all genes (inactivation – 5’ exons, minimal promoter, confirmation by expression analysis necessary!) – mutant selection in silico, ordering seeds Gene1 Gene2 Gene3 = sites of T-DNA insertions in individual lines (1-8) 1 2 3 4 5 6 7 8 …line number

18. WWW interphase http://signal.salk.edu/cgi-bin/tdnaexpress

19. Direct genetics - selection of mutants by altered phenotype agamous shootmeristemless

20. Mutant screens – phenotype, conditions, treatments, …

21. The same phenotypic change can result from different mutations „ there are numerous ways how to build up house incorrectly“ - allelic mutations – mutation in the same gene (x different g.) How to distinguish (recesive mutation) ? Crossing of homozygous mutants F1– wt = different genes (complementation) - mutant = allelic

22. Direct and reverse genetics in Arabidopsis Identification of mutation site + Tilling – „searching“ in non-characterized collection of lines by PCR and reasociation reverse direct

23. TILLING: detection of mutants with point mutations in certain gene Targeting induced local lesions in genomes Principle: chemical mutagenesis (EMS) PCR- and heteroduplex analysis-based screen Point mutations! ( changed regulation, interactions, … )

24. TILLING 1.PCR of selected sequence from DNA stocks isolated from mutant population 2.Reassociation with PCR fragment from wt plant 3.Cleavage of ss sites of heteroduplex + electrophoretic separation of end- labelled fragments

25. TILLING – strategy of screening

27. Based on genetic map 1.mapping – genetic linkage with genetic markers (necessity of dense polymorphic markers!) 2. identification of the gene - chromosom walking - sequencing (sequence comparisons) Identification/mapping of unknown (mutated) genes („with phenotype“) by cosegregation analysis

28. Genetic likage x crossing-over during meiosis Genetic likage x crossing-over during meiosis 1.Cytologic event Parental chromosomes Meiosis Without Crossing-over Gametes 1 2 3 4 Not recombinant Recombinant 2. Genetic result Parental Genotype (heterozygous Aa and Bb ) Locus A Locus B Meiosis Gametes Not recombinant ( same as parental genotype ) Recombinant ( new )

29. Basic set of genetic markers in Arabidopsis thaliana 2-3 in every chromosomal arm

30. AbAb c aBaB CC AbAb c aBaB AbAb c aBaB C AbAb c aBaB C AbAb C AbAb c aBaB Cc aBaB P1 (homozygot e ) P2 (homozygot e ) F1 (heterozygote) gamet es F2 – full linkage : AB:Ab:aB:ab 2:1:1:0 AbAb aBaB AbAb AbAb aBaB aBaB F2 – without linkage : AC:Ac:aC:ac = 9:3:3:1 A C Aa C A C A c AA ccCC A C A c A ccC aaa CC Cc ccC aa a aa A, B – full linkage! A, C – free recombination Cosegragation analysis in F2 generation

31. Segregation in F2 generation gametyXY (0.5)XyxYxy (0.5) XY (0.5) XXYY XY (0.25) XXYy XY (0.5) XxYY XY (0.5) XxYy XY (0.25) Xy XXYy XY (0.5) XXyy Xy XxYy XY Xxyy Xy (0.5) xY XxYY XY (0.5) XxYy XY xxYY xY xxYy xY (0.5) xy (0.5) XxYy XY (0.25) Xxyy Xy (0. 5) xxYy xY (0. 5) xxyy xy (0.25) (P=XXyy x xxYY, F1 = XxYy – frequency of gametes depends on the linkage) 9:3:3:1 (XY:Xy:xY:xy) x 4,75:2:2:0,25 = different chromosoms (arms) no linkage week genetic linkage Looking for strong linkage!

32. Types of genetic markers = trait with known or identifiable position in genetic map with polymorphism between parental genotypes (e.g. different ecotypes) Morphological (limited number) Molecular –DNA markers – detectable differences in DNA sequence –isozymes

33. Natural morphological variability of Arabidopsis ecotypes

34. Morphological markers Gene symbol NamePhenotypeLocation (chr. - cM) an-1angustifolianarrow leaves, crinkled siliques1-55.2 ap1-1apetalano petals1-99.3 pypyrimidine requiring white leaves, restored by pyrimidine2-49.1 er-1erectacompact inflorescence, blunt siliques2-43.5 hy2-1long hypocotylelongated hypocotyl, slender3-11.5 gl1-1glabrano trichomes3-46.2 bp-1brevipedicellusshort pedicels, siliques bent downwards, short plant 4-15.0 cer2-2eceriferumbright green stems, siliques bent downwards, short plant 4-51.9 ms1-1male sterileno siliques5-2.5 tt3-1transparent testa yellow seeds, no anthocyanin5-57.4

35. Molecular markers in Arabidopsis

36. DNA molecular markers (= usually an electrophoretic band) RFLP (Restriction fragment length polymorfism) + Southern RAPD (Random amplified polymorphism detection) AFLP (Amplified fragment length polymorphism) SSR (Simple sequence repeats) SNP (Single nucleotide polymorphism)

37. Cosegregation analysis with molecular markers Crossing of different genotypes with high polymorphism (multiple differences in markers)!!! Possibility of analysis of high number of markers at ones Which marker A,B,C,D is linked with locus R? fenotyp rfenotyp R r R Fenotyp:

38. Bulked segregant analysis Strong linkage – possibility to analyze in bulk phenotype rphenotype R R r

39. Examples of DNA molecular markers Known sequence and position in the genome RFLP (Restriction fragment length polymorfism) + Southern hybridization Unknown sequence and position (randomly visualized sequences), sequence and position determined subsequently only for those in genetic linkage with a trait RAPD (Random amplified polymorphism detection) AFLP (Amplified fragment length polymorphism)

40. RFLP

41. RAPD

42. AFLP

43. Finding of two markers surrounding mutated gene  „Chromosome walking“ Mutovaný gen X Libraries of big genomic fragments YACs, BACs = yeast (bacterial) arteficial chromosome, ~ 300 (100) kbp cosmids ( fág, 50 kbp) Looking for overlaps using hybridization

44. Marker assisted selection (MAS) Molecular marker in strong genetic linkage with certain trait can be used for screening of hybrids instead of the phenotypic characterization Advantages: Not influenced by environmental conditions Screens of seedlings Often simple and cheaper Possibility to distinguish between homo- and heterozygots (using certain markers)

45. Identification of genes by function (interaction)

46. Yeast two-hybrid screen for protein interactors