1. Non-Mendelian Genetics Unusual patterns of gene transmission Organelle Genetics Segregation distortion meiotic drive or “selfish genes” Unusual patterns of gene expression Maternal effect genes Epigenetics Small RNAs & gene silencing Imprinting Paramutation

2. Non-Mendelian Genetics - Objectives Recognize when a phenotypic or genotypic progeny ratio is non-Mendelian Determine whether there is any type of inheritance pattern in the non-Mendelian progeny (e.g. – maternal, paternal, etc.) Explain the most common mechanisms creating non-Mendelian progeny ratios Given a set of non-Mendelian observations, determine which mechanisms are consistent with the observations, and which could not explain the observations

3. Reproductive cycles of plants and animals (Walbot & Evans, Nature Rev Genet 4:369) Haploid stage is restricted to the gametes Fertilization of two gametes produces the embryo Animals establish the gamete- producing cells (germ-line) very early in development Before this zebrafish even looks like a fish, the cells producing the gametes that will create the next generation have already been established ? advantages and dis- advantages This is why environmental conditions during embryonic development can affect subsequent generation(s)

4. Reproductive cycles of plants and animals (Walbot & Evans, Nature Rev Genet 4:369) Plants establish the gamete-producing cells from somatic cells late in development Environmental cues induce meristem cells to produce floral organs instead of vegetative cells ? advantages and dis-advantages Haploid stage (gametophyte) is multicellular Dominant life stage in lower plants Double fertilization: 1 egg + 1 sperm > 2N embryo 2 central cells + 1 sperm > 3N endosperm

5. (Mosher & Melnyk Trends Plant Sci 15:204) Double fertilization in plants 1N

6. Endosymbiont origin of cellular organelles Derived from bacterial endosymbionts These became specialized for respiration and photosynthesis Molecular phylogeny mitochondria ~ to  proteobacteria plastids ~ to cyanobacteria Genome reduction many ancestral genes re-located to the nucleus

7. Organelle Genomes Small but essential! mitochondria (site of respiration) plastids (site of photosynthesis) Multiple organelles and genomes per cell 20 – 20,000 genomes per cell, depending on cell type Organized in nucleoids nucleoprotein complexes containing multiple genome copies not to be confused with nucleosomes Non-Mendelian inheritance commonly maternal “paternal leakage”, especially with interspecific crosses some organisms paternal or bi-parental

8. Organelle Genomes Necessary but insufficient for organelle function Support organelle functions membrane-associated respiratory or photosynthetic proteins Support organelle gene expression rRNAs, tRNAs and ribosomal proteins Nuclear gene products also required translated in cytosol imported into the organelle ~ 10% of nuclear genes predicted mitochondrial targeting ~15% of nuclear genes predicted plastid targeting

9. GenomePhysical map Plant plastid150 kb circle Plant mitochondria 150 – 2000 kb multipartite Human mitochondria 17 kb circle Yeast mitochondria 75 kb circle Organelle genomes Similar in coding Size differences primarily due to non-coding sequences

10. Human mitochondrial DNA map showing representative pathogenic and adaptive base substitution mutations. Wallace D C, and Chalkia D Cold Spring Harb Perspect Biol 2013;5:a021220

11. [Taylor & Turnbull Nature Rev Genet 6:391] For your reading pleasure: Comparison between human nuclear and mitochondrial genomes

12. Corens (1909) Mirabilis seed parent x pollen parent reciprocal crosses: green x variegated 100% green progeny variegated x green green, variegated & white progeny variable, non-Mendelian proportions Bauer (1909) Pelargonium seed parent x pollen parent reciprocal crosses: green x variegated variable and non-Mendelian proportions e.g. 208 green – 5 variegated – 0 white variegated x green variable and non-Mendelian proportions e.g. 83 green – 40 variegated – 3 white Inheritance of plant organelle genomes [Image - Weaver and Hedrick, 1992 Genetics, WC Brown, Dubuque] [example data from Tilney-Bassett, Heredity 20:451]

13. Some common features of organelle genome inheritance Differences in reciprocal crosses Non-Mendelian progeny ratios Somatic segregation segregation of genotypes through mitotic divisions observed as sectors of somatic tissues (e.g. green and white) Heteroplasmy cells or organisms having two (or more) organelle genotypes Homoplasmy cells or organisms having a single genotype via somatic segregation – the sorting of organelle genomes from heteroplasmy to homoplasmy through mitotic cell divisions

14. Organelle genome markers Functional markers are challenging to obtain and investigate. Why? Molecular markers are easy to develop and detect Abundance of organelle genomes in the cell RFLPs PCR-based sequence characterized amplified regions (SCARS) PCR-based cleaved amplified sequences (CAPS) A few PCR-based microsatellites Single nucleotide polymorphisms (SNPs) used extensively in humans

15. Fig. 2. Southern blot of mspI-digested DNA of parents and progeny from a reciprocal cross showing plastid DNA polymorphism. Lane 1, Falcicata; lane 2, Sativus; lanes 3- 12, progeny from the cross of Fal x Sat; lanes 13-20, progeny from the cross Sat X Fal (maternal parent listed first in crosses) Fig. 3. Southern blot of mspI-digested DNA of parents and progeny from a reciprocal cross showing mitochondrial polymorphism. Lane 1, Fal; lane 2, Sat; lanes 3-12, progeny from the cross of F x S; lanes 13-20, progeny from the cross Sat X Fal (maternal parent listed first in crosses) Organelle genome inheritance in Medicago sativa (alfalfa) (Schuman and Hancock, Theor Appl Genetics 78:863)

16. (Mosher & Melnyk Trends Plant Sci 15:204) Organelle events in plant reproduction (Nagata J Plant Res 123:193)

17. Organelle events in plant reproduction (Nagata J Plant Res 123:193) Musa accuminata (banana) has a paternal mit DNA transmission pattern Generative cell mitochondrial stained with DiOC6 (left) contain abundant DNA stained with DAPI (right) ??? But how is maternal DNA eliminated

18. Digestion of sperm mitochondrial DNA (mtDNA) post fertilization in Oryzias latipes Loss of paternal mitDNA 1 hr post fertilization PCR (above) Fluorescence label (right) mitDNA (small yellow or green dots) Lost prior to the mit structure (red) a-c pre-fertilization d-f post fertilization (Nishimura et al. Proc Natl Acad Sci USA 103:1382)

19. The human mitochondrial genome [Tuppen et al. (2009) Biochim Biophys Acta. 1797:113]

20. Mitochondrial genome markers in forensics Human mitochondrial DNA hypervariable non-coding region Differs among unrelated individuals PCR amplification and sequencing Mitochondrial DNA is abundant and resistant to degradation Marker of choice for forensic samples of limited quantity and/or exposed to environmental extremes

21. Human mitochondrial DNA map showing representative pathogenic and adaptive base substitution mutations Wallace D C, and Chalkia D Cold Spring Harb Perspect Biol 2013;5:a021220

22. The migratory history of the human mtDNA haplogroups Wallace D C, and Chalkia D Cold Spring Harb Perspect Biol 2013;5:a021220 SNPs define human mitochondrial “haplogroups” and allow tracking of human migrations Founding genotypes L and M left Africa 65- 70,000 and 48,000 years before present (YBP) Further migration and mutation expanded the mitochondrial haplogroups, some of which carry mutations adaptive for cold or warm climates

23. Human mitochondrial DNA map showing representative pathogenic and adaptive base substitution mutations Wallace D C, and Chalkia D Cold Spring Harb Perspect Biol 2013;5:a021220

24. Mitochondrial mutations in human disease Maternally inherited Affect 1 in 5,000 individuals Pathogenic mutations carried by 1 in 200 live births Affected individuals are usually heteroplasmic for the mutation – why? Primary symptoms affect neuromuscular and/or secretory tissues – why?

25. MERRF tRNALys A8344G pedigree showing variable clinical expression in association with variable mtDNA mutant heteroplasmy Wallace D C, and Chalkia D Cold Spring Harb Perspect Biol 2013;5:a021220

26. MERRF tRNALys A8344G pedigree showing variable clinical expression in association with variable mtDNA mutant heteroplasmy Wallace D C, and Chalkia D Cold Spring Harb Perspect Biol 2013;5:a021220

27. Random segregation (i.e. genetic drift - Changes in allele frequency by chance, favored by small “founder”) What governs inheritance of mitochondrial mutations in mammals? (Cree et al. Biochim Biophys Acta 1792:1097) Random, preferential replication of a subpopulation when mitochondria do begin to replicate Germ cell numbers increase but mitochondrial numbers do not

28. Selection against severe mitochondrial mutations in the germline small populations of mitochondrial DNA molecules in primordial germ cell expose severe mutations to strong selection & elimination during expansion What governs inheritance of mitochondrial mutations in mammals? [Shoubridge and Wai Science 319:914]

29. Nuclear mutations can cause mitochondrial dysfunction Why is this? e.g. Friedreich ataxia Autosomal recessive mutation Expanded GAA triplet in an intron decreases abundance of the protein product Normal gene encodes a 210 amino acid protein “Frataxin” localized to the mitochondria functions in respiratory chain assembly Knockout of yeast homolog leads to severe defect in mitochondrial respiration Affected patients have accumulated Fe in mitochondria impaired mitochondrial respiration oxidative cell damage cardiomyopathy [Campuzano et al. Science 271:1432] [Babcock et al. Science 276:1709]

30. Parent 1 mt a Parent 2 mt α progeny 1N spores chloramphenicol resistant cap-r chloramphenicol sensitive cap-s 2 : 2 mt a : mt α 4 : 0 cap-r : cap-s or 0 : 4 cap-r : cap-s chloramphenicol sensitive cap-s chloramphenicol resistant cap-r 2 : 2 mt a : mt α 4 : 0 cap-r : cap-s or 0 : 4 cap-r : cap-s Mitochondrial inheritance in Saccharomyces cerevisiae What is the inheritance pattern of the mating type marker? What is the inheritance pattern of the antibiotic resistance marker?

31. Mitochondrial inheritance in Saccharomyces cerevisiae e.g. chloramphenicol (cap) resistance (r) 1 N mating type a ; cap-r x 1 N mating type  cap-s  2N zygote nuclear a /  mitotype heteroplasmic cap-r & cap-s  mitosis (somatic segregation of mitochondrial genotypes over many mitotic divisions)  2N colonies 2 genotypes of homoplasmic cells nuclear a /  ; mitotype cap-r nuclear a /  ; mitotype cap-s  meiosis (segregation of nuclear alleles) a /  cap-r a /  cap-s  2 mt a : 2 mt  0 cap-s : 4 cap-r 4 cap-s : 0 cap-r

32. Selection for yeast mutants defective in mitochondrial respiration Genotype2 % glucose 2 % glycerol 0.1 % glucose & 2 % glycerol respiration +smalllarge respiration –smallno growthsmall “petite” Colony growth phenotype on media with: Viable because, unlike animals and plants, yeast is a facultative anaerobe!

33. Classes of petite mutants ClassDesignation nuclear (usually recessive)pet – mitochondrial point mutantmit – mitochondrial deletion mutant rho – absence of mitochondrial DNArho o “rho” was the original (pre DNA era) designation for “respiratory factor” rho = mitochondrial DNA Mitochondrial inheritance in Saccharomyces cerevisiae

34. Mitochondrial mutants in different genes can complement and recombine in the diploids after crossing! What does this tell you about the behavior of mitochondria and their genomes after mating in yeast? Mitochondrial inheritance in Saccharomyces cerevisiae Parent 1 mt a Pet + Parent 2 mt α Pet + 2N diploid zygote rho - mit - nucleus: mt a / mt α Pet+ / Pet+ mitochondria: rho- & mit - will grow on glycerol if the mit- point mutation lies outside of the rho- deletion

35. Mitochondrial mutants in different genes can complement and recombine in the diploids after crossing! Will this diploid grow on glycerol? Why or why not? Mitochondrial inheritance in Saccharomyces cerevisiae Parent 1 mt a Pet + Parent 2 mt α Pet + 2N diploid zygote rho 0 mit - nucleus: mt a / mt α Pet+ / Pet+ mitochondria: rho0 & mit -

36. Mitochondrial mutants in different genes can complement and recombine in the diploids after crossing! Will this diploid grow on glycerol? Why or why not? Mitochondrial inheritance in Saccharomyces cerevisiae Parent 1 mt a pet1 - Pet2 + Parent 2 mt α Pet1 + pet2- 2N diploid zygote + mit + mit nucleus: mt a / mt α pet1- / Pet1+ Pet2+ / pet2- mitochondria: + & +

37. Parent 1 mt + Parent 2 mt - progeny 1N spores streptomycin resistant strep-r streptomycin sensitive strep-s 2 : 2 mt + : mt - 4 : 0 strep-r : strep-s streptomycin sensitive strep-s streptomycin resistant strep-r 2 : 2 mt + : mt - 4 : 0 strep-s : strep-r Plastid inheritance in Chlamydomonas reinhardtii What is the inheritance pattern of the mating type marker? What is the inheritance pattern of the antibiotic resistance marker? How is this different from inheritance of antibiotic resistance marker we saw in yeast? image from Chlamydomonas Teaching Center http://biology.ecsu.ctstateu.edu/ChlamyTeach/chlamymain.htm

38. image from Chlamydomonas Teaching Center http://biology.ecsu.ctstateu.edu/ChlamyTeach/chlamymain.htm Plastid inheritance in Chlamydomonas reinhardtii e.g. streptomycin (strep) resistance (r) 1 N mating type + ; strep-r x 1 N mating type - ; strep-s  2N zygote nuclear mt+ / mt - plastid heteroplasmic strep-r & strep-s  meiosis (no diploid phase) (segregation of nuclear alleles) (destruction of mt- plastid genome)  2 mt+ : 2 mt - 4 strep- r : 0 strep-s

39. image from Chlamydomonas Teaching Center http://biology.ecsu.ctstateu.edu/ChlamyTeach/chlamymain.htm Growth Phenotype conditions: genotype: + acetate – light – acetate + light (requires photosynthesis) photosynthesis + (acetate +) ++ photosynthesis – (acetate –) (acetate requiring) +– Selection for Chlamydomonas mutants defective in photosynthesis Chlamydomonas is a facultative heterotroph! It can fix CO 2 by photosynthesis or use acetate instead! Acetate requiring mutants imply the strain cannot fix CO 2 by photosynthes Some acetate requiring mutants are inherited in a bi- parental (Mendelian) fashion Some acetate requiring mutants are inherited in a uniparental, mt+ fashion Why is this? These mutants are often light sensitive- why?

40. (Dent et al. Trends Plant Sci 6:365) Genetic analysis of a Chlamydomonas non-photosynthetic (acetate requiring), light-sensitive (lts) mutant mt+; lts5+ x mt -; lts5- v 2N zygote v meiosis v Two tetrads of 4 haploid progeny spores are shown, replicated on two plates exposed to different growth conditions tetrad 1 tetrad 2 What is the segregation pattern for the lts5 mutant? Is the mutation in a nuclear or a plastid gene? Which colonies would grow without acetate; which will require acetate? plate 1 dark-grown + acetate plate 2 light-grown + acetate

41. The mechanism of uniparental, mt+ inheritance in Chlamydomonas reinhardtii image from Chlamydomonas Teaching Center http://biology.ecsu.ctstateu.edu/ChlamyTeach /chlamymain.htm http://biology.ecsu.ctstateu.edu/ChlamyTeach Ruth Sager model: based upon bacterial restriction / modification systems mt- plastid DNA digested in the zygote mt+ plastid DNA modified & protected Recent work supports this model: nuclease localized to the mt- plastids in young zygotes (Nishimura et al. Genes & Development 16:1116) mt- plastid DNA is preferentially degraded in young zygotes (Nishimura et al. Proc Natl Acad Sci USA 96:12577) mt+ plastid DNA is preferentially methylated prior to mating (Nishiyama et al. Proc. Natl. Acad. Sci. USA 99:5925)