Mendel and Inheritance MUPGRET Workshop June 17, 2004

Genetic variation In the beginning geneticists studied differences they could see in plants. These differences are called morphological differences. Individual variants are referred to as phenotypes, ex. tall vs. short plants or red vs. white flowers.

Trait A broad term encompassing a distribution of phenotypic variation. Example: Trait: Disease resistance Phenotype: resistant vs. susceptible Morphological differences associated with the trait might include fungal infection, fungal growth, sporulation, etc.

Mendel Monk at the St. Thomas monastery in the Czech Republic. Performed several experiments between 1856 and 1863 that were the basis for what we know about heredity today. Used garden peas for his research. Published his work in 1866.

Mendel Results are remarkably accurate and some have said they were too good to be unbiased. His papers were largely ignored for more than 30 years until other researchers appreciated its significance.

Garden Pea Pisum sativum Diploid Differed in seed shape, seed color, flower color, pod shape, plant height, etc. Each phenotype Mendel studied was controlled by a single gene.

Terms Wild-type is the phenotype that would normally be expected. Mutant is the phenotype that deviates from the norm, is unexpected but heritable. Notice that this definition does not imply that all mutants are bad in fact many beneficial mutations have been selected by plant breeders.

Advantages of plants Can make controlled hybrids. Less costly and time consuming to maintain than animals. Can store their seed for long periods of time. One plant can produce tens to hundreds of progeny.

Advantages of plants Can make inbreds in many plant species without severe effects that are typically seen in animals. Generation time is often much less than for animals. Fast plants (Brassica sp.) Arabidopsis

Principle of Segregation Parental Lines Round Wrinkled X All round F 1 progeny Self-pollinate Round 5474 Wrinkled Round : 1 Wrinkled

Mendel’s Results Parent Cross F 1 Phenotype F 2 data Round x wrinkled Round 5474 : 1850 Yellow x green Yellow 6022 : 2001 Purple x white Purple 705 : 224 Inflated x constricted pod Inflated 882 : 299 Green x yellow pod Green 428 : 152 Axial x terminal flower Axial 651 : 207 Long x short stem Long 787 : 277

Important Observations F 1 progeny are heterozygous but express only one phenotype, the dominant one. In the F 2 generation plants with both phenotypes are observed  some plants have recovered the recessive phenotype. In the F 2 generation there are approximately three times as many of one phenotype as the other.

3 : 1 Ratio The 3 : 1 ratio is the key to interpreting Mendel’s data and the foundation for the the principle of segregation.

The Principle of Segregation Genes come in pairs and each cell has two copies. Each pair of genes can be identical (homozygous) or different (heterozygous). Each reproductive cell (gamete) contains only one copy of the gene.

Principle of Segregation Either copy of the gene is equally likely to be included in a gamete. One male and one female gamete combine to generate a new individual with two copies of the gene.

Allele One of two to many alternative forms of the same gene (eg., round allele vs. wrinkled allele). Alleles have different DNA sequences that cause the different appearances we see.

Mendel’s Principle of Segregation In the formation of gametes, the paired hereditary determinants separate (segregate) in such a way that each gamete is equally likely to contain either member of the pair.

Principle of Segregation Parental Lines Round (WW) Wrinkled (ww) X All round F 1 progeny (Ww) Self-pollinate Round (WW + Ww) 5474 Wrinkled (ww) Round : 1 Wrinkled

Punnett Square A (½)a (½) A (½)AA (½ x ½ = ¼) Aa (½ x ½ = ¼) a(½)Aa (½ x ½ = ¼) aa (½ x ½ = ¼) Male Female ¼ AA :½ Aa : ¼ aa

Round vs. wrinkled The SBEI causes the round vs. wrinkled phenotype. SBEI = starch-branching enzyme Wrinkled peas result from absence of the branched form of starch called amylopectin. When dried round peas shrink uniformly and wrinkled do not.

Round vs. wrinkled The non-mutant or wild-type round allele is designated W. The mutant, wrinkled allele is designated w. Seeds that are Ww have half the SBEI of wild-type WW seeds but this is enough to make the seeds shrink uniformly. W is dominant over w.

Round vs. wrinkled An extra DNA sequence is present in the wrinkled allele that produces a non- functional SBEI and blocks the starch synthesis pathway at this step resulting in a lack of amylopectin.

A Molecular View ParentsF1F1 F 2 Progeny WW ww Ww¼WW ¼Ww ¼wW ¼ww 1: 2 : 1 Genotype = 3: 1 Phenotype

Chi-Squared Analysis Tests if your observations are statistically different from your expectation. For example does the Mendel data fit the 3:1 hypothesis? Chi-squared =  [(observed-expected) 2 /expected]

Testcross and Backcross x x WWww Ww ww Ww ww Ww wWwww wWwww Parents F1F1 Testcross Progeny

Mendel and two genes x Round Yellow Wrinkled Green All F1 Round, Yellow Round Yellow 315 Round Green 108 Wrinkled Yellow 101 Wrinkled Green 32

Mendel and two genes Round Yellow 315 Round Green 108 Wrinkled Yellow 101 Wrinkled Green 32 Round = 423 Wrinkled = 133 Yellow = 416 Green = 140 Each gene has a 3 : 1 ratio.

Punnett Square Yellow ¾ Green ¼ Round ¾ Round, Yellow ¾ x ¾ = 9/16 Round, Green ¾ x ¼ = 3/16 Wrinkled ¼ Wrinkled, Yellow ¼ x ¾ = 3/16 Wrinkled, Green ¼ x ¼ = 1/16

Ratio for a cross with 2 genes Crosses with two genes are called dihybrid. Dihybrid crosses have genetic ratios of 9:3:3:1.

Principle of Independent Assortment Ww Gg WG ¼ Wg ¼ wG ¼ wg ¼ F1F1 Gametes & Frequencies If a gamete contains W the probability that it contains G is equal to the probability that it contains g.

¼ WG¼ Wg¼ wG¼ wg ¼ WGWW GG 1/16 WW Gg 1/16 WwGG 1/16 WwGg 1/16 ¼ WgWWGg 1/16 WWgg 1/16 WwGg 1/16 Wwgg 1/16 ¼ wGWwGG 1/16 WwGg 1/16 wwGG 1/16 wwGg 1/16 ¼ wgWwGg 1/16 Wwgg 1/16 wwGg 1/16 wwgg 1/16

Phenotypes W = Round w = Wrinkled W is dominant to w. G = Yellow g = Green G is dominant to g.

F 2 Progeny GenotypePhenotype 1/16 WWGG + 2/16 WWGg + 2/16 WwGG + 4/16 WwGg 9/16 Round Yellow 1/16 wwGG + 2/16 wwGg 3/16 Wrinkled Yellow 1/16 WWgg + 2/16 Wwgg 3/16 Round Green 1/16 wwgg 1/16 Wrinkled Green

Principle of Independent Assortment Segregation of the members of any pair of alleles is independent of the segregation of other pairs in the formation of reproductive cells.

Summary of Mendel Inherited traits are controlled by the alleles present in the reproductive cells that fuse to form the embryo. In a diploid, progeny inherit one allele from the mother and one from the father. Differences in the DNA sequence of two alleles for a gene may result in different phenotypes.

Summary The phenotype is the same if the gene is inherited from the mother or from the father. One allele from the diploid is inherited in each reproductive cell.

ESTs Defined EST is an acronym for Expressed Sequenced Tags. cDNAs with map locations. ESTs represent the genes active in a particular tissue at specific stage of development. The cellular function of an EST can often be derived by comparison to sequence databases.

ESTs are important markers for physical mapping. EST mapping, coupled with trait mapping, will help link genes to phenotypes. ESTs are the basic building blocks for high density “gene chips”. EST Mapping: Utility

Efficient Markers for Maize Simple Sequence Repeats(SSRs) Develop SSR markers Map SSRs genetically Use SSRs to anchor physical map to genetic map. Use SSRs to map genes for traits more efficiently.

SSRs: What and how? Dinucleotide and trinucleotide repeats. ex. CACACACACA Different corn lines have different numbers of repeats. (CA) 10, (CA) 14 These differences can be seen on a high resolution agarose gel. Can be used for mapping.

SSR Mapping C A T G

Scoring data A B H H A B A H

Recombination and Mapping P Assume that the frequency of crossing- over is equal along the chromosome. P Two genes that are very close to one another will have a lower likelihood of having a cross-over between them than two genes that are very far apart.

Recombination and Mapping P So, we can determine the relative distance between genes by counting the number of recombinant genotypes for each pair of genes. ê Lots of recombinants = far apart ê Few recombinants = close together

BHBBAHBHHBHHBHB umc157 HHBBABBHHBBBBAB umc76 BHBBABHAHHBHBAB asg45 BHBBABBAHHBHBAB zb4 BHBBHBHAHHBHBAB csu3 How Maximum Likelihood Works

BHBBAHBHHBHHBHB umc157 BHBBABHAHHBHBAB asg45 HHBBABBHHBBBBAB umc76 BHBBABBAHHBHBAB zb4 BHBBHBHAHHBHBAB csu3

Outline of mapping Sequence cDNAs. Analyze sequences for SSRs. Check for polymorphisms between parents. Collect mapping population data for polymorphic primers. Construct genetic map.

Screening

Workshop Outline Set up PCR reactions (Thursday afternoon). Load gels and electrophorese (Friday morning). Photograph gels, collect genotype data (Friday afternoon).