Making the Intermated B73 X MO17 mapping population of recombinant inbred lines (RILs)

1. Making the Intermated B73 X MO17 mapping population of recombinant inbred lines (RILs) Mo17 B73 X Single F2 plant was selfed

2. X Grow up 200 plants, random mating Illustration of the genotype of 6 of those 200 plants. Recombination during meiosis results in the exchange of chromosome segments

3. x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x Select 100 ears, pick 5 kernels from each ear. Put in a bag, shake, plant, more random matings (2nd generation). Repeat, (repeat….) From these lines, generate Recombinant Inbred lines by repeated selfing (5x or more) And Tahdah!… the Recombinant Inbred mapping population

4. Finding quantitative trait loci (QTLs) You test your population of plants for the presence of a phenotype (e.g. leaf size) and you determine the statistical likelihood that each molecular marker is associated with the trait. This gives you a LOD score (logarithm of the odds that they are associated). You plot LOD scores along the chromosome, and where it is highest you have a QTL locus (one or more genes) that determines your phenotype. In the example shown, Antirrhinum majus (snapdragon) has 4 QTLs for leaf size.

5. How do we get pairing of homoeologous chromosomes? “Wild sex” in this example means inter- specific crossing or hybridization. Homologous chromosomes readily pair during meiosis, but homoeologous Chromosomes do not. “Homoeologous” means “partially homologous”, usually indicating some original ancestral homology. Pairing is prevented by Certain genes that evolve as part of speciation. Can those genes be identified and then knocked out? What we need is a little more

6. Breaking the cereal species barrier:pairing of homoeologous chromosomes Bread wheat contains three closely related genomes: A, B and D. Chromosome pairing during meiosis is confined to strict homologues, despite the co-existence in the genome of homoeologous chromosomes. This control maintains the integrity of the three genomes. The genetic control of chromosome pairing in wheat is dependent on a series of genes that suppress and promote homoeologous pairing (Ph genes). The strongest effect on pairing is associated with a gene (or genes) at the Ph1 locus) that suppresses homoeologous chromosome pairing within the polyploid wheat genome. In the absence of Ph1, homoeologous recombination can occur between wheat chromosomes and those from related species or genera. An X-ray-induced deletion at the Ph2 locus, ph2a, and a chemically induced mutant, ph2b, have demonstrated that removal of the Ph2 gene induces an intermediate level of homoeologous chromosome pairing in wheat hybrids with alien species but does not affect chromosome pairing in wheat itself. These observations suggest that Ph2 will be a valuable resource for the introgression of alien genes from related species into bread wheat, with minimum disruption of endogenous homologous chromosome pairing.

7. Rice flower 1. Put pollen from Oryza glaberrima on the pistil of Oryza sativa, and allow embryo to develop. Collect the seeds that are formed with very low frequency. Male stamens 2. Backcross the progeny multiple times to O. sativa so that you end up with a plant that is 85 % Sativa and 15 % Glaberrima. Female pistil AA X BB A (pollen) + B (egg) --> A (50%)B (50%) Mostly infertile and very few seeds are formed After back-crossing --> A(85%)B(15%) highly fertile plant Such a hybrid does not form in Nature, first because the two species grow on different continents, but second because the pollination is nearly infertile. It combines genes from two different species, but is not a GMO.

8. Inter-specific hybridization: Nerica, an upland rice for Africa. Asian rice African rice Nerica rice Oryza sativa (Asian upland rice): non-shattering, resistant to lodging, high yield potential Oryza glaberrima (African rice): drought tolerant, disease resistant, weed-suppressing Nerica rice combines the best of both species.

9. “Wide” crossing of wheat and rye requires embryo rescue and chemical treatment to double the number of chromosomes. Triticale http://gnome.agrenv.mcgill.ca/breeding/tritical.htm

10. Triticale, a new cereal created in the lab.Triticale, a cross between wheat and rye, was produced by embryo rescue of the product of fertilization and a chemically induced doubling of the chromosomes. Embryo rescue becomes necessary when fertile offspring is never produced by an interspecific cross. Triticale is now grown all over the world Wheat, triticale and rye

11. “Soluble solids” are the holy grail of the tomato processing industry, because the most expensive part of making tomato sauce/paste is evaporating the water. Useful yield is determined by soluble solids. Roma tomatoes have high soluble solids. Roma tomatoes have less water Refractometer to measure BRIX value Plant breeders and genetic engineers focus on “soluble solids” (sucrose and other soluble molecules), which are measured as the BRIX value of the tomato juice.

12. Make recombinant inbred lines (RILs) and identify quantitative trait loci (QTLs) for Brix value. Zamir and colleagues at the Hebrew University made RILs between Solanum lycopersicum (domestic tomato) and Solanum pennellii. They identified 25 QTLs for soluble solids measured as “Brix value”. One QTL improved Brix by 10-20%. Mapping showed it to map to a chromosome region that encodes a fruit specific invertase. Fine mapping narrowed it down to a SNP in a 484 bp region of the invertase. In one specific place S.pennellii has a D (Asp) - in common with invertases from carrot, tobacco, potato and pea, but the S. lycopersicum invertase has an E (Glu)The enzyme has a higher affinity (lower Km) for sucrose and is more efficient at unloading sucrose in the fruit.Science, 305: 1786-9 (2004) Glu Asp Brix = refractive index and measures mostly sugars.