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

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B73 Mo17

Single F2 plant was selfed

XGrow 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

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)

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And Tahdah!… the Recombinant Inbred mapping population

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.

Finding quantitative trait loci (QTLs)

What we need is a little more

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?

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.

Male stamens

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

Rice flower

2. Backcross the progeny multipletimes to O. sativa so that you end up with a plant that is 85 % Sativa and 15 % Glaberrima.

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.

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1. Put pollen from Oryza glaberrimaon the pistil of Oryza sativa, andallow embryo to develop. Collect the seeds that are formed with very low frequency.

Inter-specific hybridization: Nerica, an upland rice for Africa.

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.

African rice

Nerica rice

Asian rice

http://gnome.agrenv.mcgill.ca/breeding/tritical.htm

“Wide” crossing of wheat and rye requires embryo

rescue and chemical treatment to double the

number of chromosomes.

Triticale

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.

Wheat, triticale and ryeTriticale is now grown all over the world

“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.

Refractometer to measure BRIX value Plant breeders and genetic engineers focus on

“soluble solids” (sucrose and other soluble molecules), which are measured as the BRIXvalue of the tomato juice.

Roma tomatoes have less water

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)

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Make recombinant inbred lines (RILs) and identify quantitative trait loci (QTLs) for Brix value.

Glu

Asp

Brix = refractive index and measuresmostly sugars.