Genotyping and confirmation of the Candy Leaf ( ) gene mutation after introgressions with other breeds of corn

Cesar Morfin Sarah Hake

Abstract

Results/Discussion

Methods

The project called Candy Leaf ( CAL )

involves improving biofuel feed stocks.

Biofuels will be using the cell walls which

are made of 6 and 5 carbon sugars. This

particular type of maize has a mutation in

a gene that creates an increased amount

of glucose in the cell walls in comparison

to the average maize plant. This property

makes the CAL plant ideal as an improvement over already available biofuels. However, there is a difficulty in the identification of the CAL mutation in maize feedstock since it lacks a visible phenotype. This difficulty arises as we breed the mutation into different lines of corn.

DNA Preps for Genotyping Introgressions - Extract Maize tissue from the youngest tissue and sort into 1.5ml tubes. - Load 5 3.2 mm chrome beads into each sampled filled tube, then add 500ul of extraction buffer to each - Load samples on to the Qiagen adapter, then load on to the bead beater for 2.5 minutes with the frequency set at 30/s - Add 35ul of SDS and vortex briefly then add 139ul of ice cold KAC and invert several times. - Centrifuge for 15 min at a speed <16k rpm - Transfer supernatant (about 500ul) to a new tube then add 350ul of isopropanol. - Centrifuge for 10 min @ max speed then discard supernatant and add 100ul of 70% EtOH. - Spin @ max speed for 5 min and invert to dry, once dried out add 100ul of TE w/ 1% Rnase. - Loaded 1ul for PCR reaction

BNLG 1702 UMC 2141

Methods cont. Confirmation of CAL gene mutation Step 1. Tissue Extraction - Prepare a PCR mixture for each primer (blng 1702 and umc 2141) and collect Maize leaves. - Obtain a .5mm tissue corer and place leaf on a hard, flat surface. Take core sample from leaf. - Place the tip of the tissue corer into PCR mixture and press the plunger to watch core enter the reaction mixture. - After each core is taken, clean the corer by moving the plunger up and down several times in 2% bleach. Then wipe the tip gently w/ a Kimwipe. Step 2. Gel Electrophoresis w/ different primers - Prepare gel using a 3.5% metaphor mixture and run for ~60 min.

After genotyping the introgressions made we can see that the CAL mutation is segregating when bred into the other lines of corn. Furthermore, the family is segregating in a 1:2:1 pattern that is expected in a Punnett square.

In reviewing the Gels in the 2nd experiment we were able to isolate some recombinants that enabled us to confirm that the CAL mutation was present .

I

- Genotyping Introgressions of the CAL mutation into other lines of corn including the brown midrib mutants which are defective in lignin.

(Families 9498 ,9499, 9500, and 9519).

- Identifying recombinants to confirm the mutant is the CAL mutation.

Objectives

Heterozygous CAL Wild Type

Figure 1. At 5 weeks old there is no visible phenotype when comparing the CAL and Wild Type plants.

Figure 4. Genotyping family 9519 Samples 1-11 were cross checked with known controls. Samples 12-16 were the controls in numerical order: CAL, B73, MO17, and 9072-4 4121

Figure 2.Degestibility and lignin content of Cal mutant. While digestibility increased w/ the CAL mutation, lignin was not affected. Data provided by Florian Kraemer .

Figure 3. Recombinant Map of cal mutant places it to a specific location in the chromosome. BNLG 1702 and GK 3 were used in the experiment.

Figure 5. chromosome 6 houses the gene of interest and also shows the location of the primers being used.