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 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 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

19. Duplicate Recessive Epistasis
AAbb x
aaBB
Low Cyanide
Low Cyanide F1
AaBb
High Cyanide AB Ab aB ab
AABB
AABb
AaBB
AaBb
AAbb
Aabb
aaBB
aaBb
aabb 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. Example: Fruit color in summer squash (Cucurbita pepo)
Dominant Epistasis
Example: Fruit color in summer squash (Cucurbita pepo) x
P1 = white fruit
P2 = yellow fruit
F1= yellow fruit F2
12 white: yellow: green

22. Example: Fruit colour in summer squash (Cucurbita pepo)
Dominant Epistasis
Example: Fruit colour in summer squash (Cucurbita pepo)
WWyy x
wwYY
White Fruit
Yellow Fruit
WwYy F1
White Fruit WY Wy wY wy
WWYY
WWYy
WwYY
WwYy
Wwyy
wwYY
wwYy
wwyy 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
Gene Interaction
Control Pattern
A-B-
A-bb
aaB-
aabb
Ratio
Additive
No interaction between loci 9 3 1
9:3:3:1
Duplicate Recessive
Dominant allele from each locus required
9:7
Duplicate
Dominant allele from each locus needed
9:6:1
Recessive
Homozygous recessive at one locus masks second
9:3:4
Dominant
Dominant allele at one locus masks other
12:3:1
Dominant Suppression
Homozygous recessive allele at dominant suppressor locus needed
13:3
Duplicate Dominant
Dominant allele at either of two loci needed
15:1

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

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
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
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
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
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. the number of genes determining the trait
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