Gene regulation Genetically related genotypes with striking phenotypic differences, but - PowerPoint Presentation

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 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

Gene regulation Focus on miRNA, siRNA

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. RNAi

Complete dominance : AA = Aa > aa.Simplest model: the functional allele vs. the non-functional allele. Deletion, altered transcription, altered translation 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) Allelic relationships at a locus Collard et al. 2005. Euphytica . Deletion P1 P2

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. Allelic Relationships at a locus

Codominance: Example: Hazelnut Allelic Relationships at a locus 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

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. Allelic Relationships at a locus Collard et al. 2005. Euphytica . P1 P2

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

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

Heterosis Mid-parent heterosis F1 > (P1+P2)/2 High parent heterosis F1 > P1; Aa >AA> aaPerhaps most useful

Cause(s) of HeterosisOver-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

The molecular basis of heterosis involves Structural variation:SNPs and INDELsSV (structural variation) C N V (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”

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

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. Allelic Variation - revisited

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. Intra-locus interactions

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

AAbb aaBB x AaBb AB Ab aB ab AB AABB AABb AaBB AaBb Ab AABb AAbb AaBb Aabb aB AaBB AaBb aaBB aaBb ab AaBb Aabb aaBb aabb Low Cyanide Low Cyanide High Cyanide F1 F2 9 High : 7 Low Cyanide Duplicate Recessive Epistasis Remember: Doubled Haploid Ratio Identical phenotypes are produced when either locus is homozygous recessive ( A_bb ; aaB _), or when both loci are homozygous recessive ( aabb ).

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 Duplicate Recessive Epistasis

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

Example: Fruit colour in summer squash (Cucurbita pepo) Dominant Epistasis WWyy wwYY x WwYy WY Wy wY wy WY WWYY WWYy WwYY WwYy Wy WWYy Wwyy WwYy Wwyy wY WwYY WwYy wwYY wwYy wy WwYy Wwyy wwYy wwyy White Fruit Yellow Fruit White Fruit F1 F2 A dominant allele at the W locus suppresses the expression of any allele at the Y locus

Gene Interaction Control Pattern A-B- A-bb aaB - aabb Ratio Additive No interaction between loci 9 3 3 1 9:3:3:1 Duplicate Recessive Dominant allele from each locus required 9 3 3 1 9:7 Duplicate Dominant allele from each locus needed 9 3 3 1 9:6:1 Recessive Homozygous recessive at one locus masks second 9 3 3 1 9:3:4 Dominant Dominant allele at one locus masks other 9 3 3 1 12:3:1 Dominant Suppression Homozygous recessive allele at dominant suppressor locus needed 9 3 3 1 13:3 Duplicate Dominant Dominant allele at either of two loci needed 9 3 3 1 15:1 Dihybrid F2 ratios with and without epistasis

Gene Interaction Control Pattern AABB AAbb aaBB aabb Ratio Additive No interaction between loci 1 1 1 1 1:1:1:1 Duplicate Recessive Dominant allele from each locus required 1 1 1 1 1:3 Duplicate Dominant allele from each locus needed 1 1 1 1 1:2:1 Recessive Homozygous recessive at one locus masks second 1 1 1 1 1:1:2 Dominant Dominant allele at one locus masks other 1 1 1 1 2:1:1 Dominant Suppression Homozygous recessive allele at dominant suppressor locus needed 1 1 1 1 3:1 Duplicate Dominant Dominant allele at either of two loci needed 1 1 1 1 3:1 Dihybrid doubled haploid ratios with and without epistasis

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

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

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

A model for intra-locus repression and expression

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

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

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

Making (near) isogenic lineshttp://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!

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.

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). 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? 17 predicted genes

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

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

The facultative option Hard-wired for low temperature tolerance and short-day sensitivityNo vernalization sensitivity The option to fall-plant and/or spring-plant the same variety Reduces risk Maximizes opportunities Streamlines seed production and end-use

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?

The facultative option Hard-wired for low temperature tolerance and short-day sensitivityNo 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, ….., ……….

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).

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

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