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Gene regulation

Gene regulation. Genetically related genotypes with striking phenotypic differences, but similar allelic architecture. Within a genotype – striking phenotypic differences between growth stages and/or between tissues. Gene regulation.

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Gene regulation

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  1. Gene regulation Genetically related genotypes with striking phenotypic differences, but similar allelic architecture. Within a genotype – striking phenotypic differences between growth stages and/or between tissues.

  2. Gene regulation • Promoters - Efficiency, constitutive, tissue-specific, inducible: • CaMV 35S, Glutelin GT1, Cis-Jasmone • Transcription factors - Facilitate, enhance, repress: • Nud, Vrs1 • mRNA stability - minutes to months: • 5’cap, 3’tail • Chromatin remodeling: • Accessibility of DNA to transcription machinery. • RNAi: • hnRNA, lncRNA, miRNA, puRNA, shRNA, snoRNA, siRNA, tiRNA,….,…. • Translational and post-translational modification of proteins: • Protein synthesis rate, transport, stability, activity

  3. Gene regulation Focus on miRNA, siRNA

  4. RNAi • Regulation of mRNA via: • mRNA cleavage: RISC pairs with target, Slicer enzyme cuts mRNA, mRNA pieces degrade. • Translation inhibition: miRNA inhibits translation by binding with target mRNA. • Transcriptional silencing: siRNA silences transcription through chromatin alteration. • mRNA degradation: Slicer-independent siRNA + protein.

  5. Allelic relationships at a locus • Complete dominance: AA = Aa > aa. • Simplest model: the functional allele vs. the non-functional allele. • Deletion, altered transcription, altered translation

  6. Allelic relationships at a locus • Complete dominance with molecular markers: BB = Bb. No bb. • Not ideal, since cannot distinguish Bb heterozygotes from BB homozygotes. • Simplest model: The target DNA sequence is there (BB (twice) or Bb (once) or is not there (bb). • Mechanisms: • Deletion, (insertion) Deletion P2 P1 Collard et al. 2005. Euphytica.

  7. Allelic Relationships at a locus • Incomplete (partial) dominance: • Example: Red x White gives a pink F1. The F2 phenotypes are 1 Red: 2 Pink: 1 White. • Explanation: Red pigment is formed by a complex series of enzymatic reactions. Plants with the dominant allele at the I locus produce an enzyme critical for pigment formation. Individuals that are ii produce an inactive enzyme and thus no pigment. In this case, II individuals produce twice as much pigment as Ii individuals and ii individuals produce none. The amount of pigment produced determines the intensity of flower color.

  8. Allelic Relationships at a locus Codominance: Example: Hazelnut • One S-locus, 33 alleles • Co-dominance in stigmas (equal expression of both alleles) • Dominance or co-dominance in pollen • If the same allele is expressed by the stigma and the pollen, the cross is incompatible

  9. Allelic Relationships at a locus • Co-dominance with molecular markers: AA, Aa, aa • Ideal: can distinguish Aa heterozygotes from AA homozygotes. • Simplest model: The target DNA sequences at the two alleles are there. • Deletion, insertion. P1 P2 Collard et al. 2005. Euphytica.

  10. Allelic Relationships at a locus Overdominance: Aa >AA, aa • Cross two inbred parents: • The F1 deviates significantly from the “high” parent. • Possible explanation of heterosis (hybrid vigor)

  11. Overdominance and hybrid vigor (heterosis) Single locus Model P2 Mid-Parent P1 F1 aa m AA Aa Phenotype

  12. Heterosis Mid-parent heterosis • F1 > (P1+P2)/2 High parent heterosis • F1 > P1; Aa>AA>aa • Perhaps most useful

  13. Cause(s) of Heterosis • Over-dominance: • Heterozygote advantage: Aa > AA • F1’s always better than inbreds • Dispersed dominant genes theory: • Phenotype controlled by several (many) genes • Remember quantitative inheritance • Favourable alleles dispersed amongst parents • (++/++/++/--/--/ x --/--/--/++/++/ = F1 +-/+-/+-/+-/+-) • Implication: • Should be able to develop inbreds = F1 • Implications for vegetative and/or apomictic propagation of hybrids

  14. The molecular basis of heterosis involves • Structural variation: • SNPs and INDELs • SV (structural variation) • CNV (copy number variation) • PAV (presence/absence variation) • Differences in expression level: • Parents – differential expression of most genes • F1 • mid-parent level of gene expression • Non-additive expression • Epigenetics: • At the time of writing, “potential and possibilities”

  15. The molecular basis of heterosis Conclusions: No simple, unifying explanation for heterosis: specificity at the species, cross, trait levels Extensive functional intra-specific variation for genome content and expression Heterosis generally the result of the action of multiple loci: quantitative inheritance

  16. Allelic Variation - revisited • Many alleles are possible in a population, but in a diploid individual, there are only two alleles possible at a locus. • Remember polyploids. • Mutation is the source of new alleles. • Remember transgenics and edits. • There are many levels of allelic variation: • DNA sequence changes with/without changes in phenotype. • Differences in phenotype due to effects at the transcriptional, translational, and/or post-translational levels. • Remember epigenetics.

  17. Intra-locus interactions Epistasis: Interaction(s) between alleles at different loci Remember: Gene interactions are the rules rather than the exceptions. Example: Duplicate recessive epistasis: Cyanide production in white clover.

  18. Duplicate Recessive Epistasis Parental, F1, and F2 phenotypes: Parent 1            x           Parent 2 low cyanide low cyanide F1 F2 (9 high cyanide : 7 low cyanide) high cyanide https://bulawayo24.com/index-id-news-sc-national-byo-101389.html

  19. Duplicate Recessive Epistasis AAbb x aaBB Low Cyanide Low Cyanide AaBb F1 High Cyanide F2 9 High: 7 Low Cyanide • Identical phenotypes are produced when either locus is homozygous recessive (A_bb; aaB_), or when both loci are homozygous recessive (aabb). Remember: Doubled Haploid Ratio

  20. Duplicate Recessive Epistasis Precursor Enzyme 1 (AA; Aa)    Glucoside Enzyme 2 (BB; Bb) Cyanide If Enzyme 1 = aa; end pathway and accumulate Precursor; if Enzyme 2 = bb; end pathway and accumulate Glucoside

  21. Dominant Epistasis Example: Fruit color in summer squash (Cucurbitapepo) x P2 = yellow fruit P1 = white fruit F1= yellow fruit F2 12 white: 3 yellow: 1 green

  22. Dominant Epistasis Example: Fruit colour in summer squash (Cucurbitapepo) WWyy x wwYY White Fruit Yellow Fruit WwYy F1 White Fruit F2 A dominant allele at the W locus suppresses the expression of any allele at the Y locus

  23. Dihybrid F2 ratios with and without epistasis

  24. Dihybrid doubled haploid ratios with and without epistasis

  25. Vernalization sensitivity and cold tolerance in barley Epistasis, near-isogenic lines, genotyping, sequencing, phenotyping, epigenetics, and climate change

  26. The phenotype: Vernalization requirement/sensitivity • In winter growth habit genotypes, exposure to low temperatures necessary for a timely transition from the vegetative to the reproductive growth stage. • Why of interest? • Flowering biology = productivity (yield) • Correlated with low temperature tolerance • Low temperature tolerance require for winter survival • Many regions have winter precipitation patterns • Fall-planted, low temperature-tolerant cereal crops - a tool for dealing with climate change

  27. The genotype: Vernalization requirement/sensitivity • Three-locus epistatic interaction: VRN-H1, VRN-H2, VRN-H3 7:1 ratio (Doubled haploid) Takahashi and Yasuda (1971)

  28. A model for intra-locus repression and expression

  29. Vernalization sensitivity and low temperature tolerance • Low temperature tolerance • Fr-H1 • Alternative functional alleles • Fr-H2 • CBF gene family and CNV • Fr-H3 • Unpublished candidate gene • Vernalization • VRN-H1 • Alternative functional alleles • Chromatin remodeling • VRN-H2 • Gene duplication and deletion • VRN-H3 • Alternative functional alleles • Copy number variation

  30. GWAS for quantitative traits Low temperature tolerance (winter survival) Vernalization sensitivity Winter survival Vernalization sensitivity (Publication in preparation)

  31. Understanding the germplasm that Takahashi and Yasuda created using: • SNP genotypes of parents and (near) isogenic lines - in linkage map order • The barley genome sequence • Gene expression patterns of specific genes • Low temperature tolerance and vernalization sensitivity phenotypic data

  32. Making (near) isogenic lines http://themadvirologist.blogspot.com/2017/01/what-is-isogenic-line-and-why-should-it.html Takahashi and Yasuda created the barley vernalization isogenic lines with 11 backcrosses and only phenotypic selection for the target alleles!

  33. Where are the introgressions and how extensive are they? Graphical SNP genotypes for the single locus VRN isogenic lines Blue = recurrent parent; red = donor parent ; pink = monomorphic SNPs • Map-ordered SNPs reveal defined introgressions on target chromosomes. • Estimates of genetic (5 – 30 cM) and physical (7 – 50 Mb) sizes of introgressions. • Alignment with genome sequence allows estimates of gene number and content within introgressions.

  34. Is VRN-H2 necessary for low temperature tolerance? Gene annotations for the VRN-H2 genes present in the winter parent and absent in the spring donor (deletion allele). 17 predicted genes • No flowering time or low temperature tolerance–related genes in the VRN-H2 introgression. • Can we therefore have the VRN-H2 deletion and maintain cold tolerance?

  35. No significant loss in low temperature tolerance with the VRN-H2 deletion

  36. VRN allele architecture, vernalization sensitivity and • low temperature tolerance Winter growth habit Facultative growth habit Takahashi and Yasuda (1971) Cuesta-Marcos et al. (2015)

  37. The facultative option Hard-wired for low temperature tolerance and short-day sensitivity No vernalization sensitivity • The option to fall-plant and/or spring-plant the same variety • Reduces risk • Maximizes opportunities • Streamlines seed production and end-use

  38. Facultative growth habit – ready for • THE CHANGE? • “Just say no” to vernalization sensitivity with the “right” VRN-H2 allele • A complete deletion • “Just say yes” to short day photoperiod sensitivity with the “right” photoperiod sensitivity allele (PPD-H2) • “Ensure” a winter haplotype at all low temperature tolerance loci • Fr-H1, FR-H2, and FR-H3 plus…. a continual process of discovery • Remember: Transgenics? Gene editing?

  39. The facultative option Hard-wired for low temperature tolerance and short-day sensitivity No vernalization sensitivity • The option to fall-plant and/or spring-plant the same variety • Take precautionary measures to maximize genetic diversity, or else….the green bridge brings on Learn the lessons of the T cytoplasm, the Cavendish banana, ….., ……….

  40. The genetic status (degree of homozygosity) of the parents will determine which generation is appropriate for genetic analysis and the interpretation of the data (e.g. comparison of observed vs. expected phenotypes or genotypes).

  41. The degree of homozygosity of the parents will likely be a function of their mating biology, e.g. cross vs. self-pollinated.

  42. Expected and observed ratios in cross progeny will be a function of: • the degree of homozygosity of the parents • the generation studied • the degree of dominance • the degree of interaction between genes • the number of genes determining the trait

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