The material presented here is based upon work supported by the National Science Foundation under Award No. EEC-0813570. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

Katelyn Hockemeyer, Tes Posekany, Miriam Lopez, Marna Yandeau-Nelson, and Nick Lauter

Lipids within and on the surface of plant cuticles have been linked to how the plant protects itself from both

environmental stress and pests, yet many of the genes responsible for lipid production and regulation remain

unknown. By studying the statistical associations between genomic loci and lipid production using QTL mapping,

four genomic loci important for surface lipid production have been targeted for further research. Each genomic

locus of interest contains millions of nucleotides and hundreds of genes. A parental backcross plant breeding

approach is being pursued to narrow down the list of candidate genes associated with surface lipid production.

INTRODUCTION

DNA Extraction

Tissue Collection

DNA ExtractionDuring the process of DNA extraction, cellular components such as the cell wall are first

broken down and separated via treatments with chemicals such as CTAB and chloroform.

After this, the DNA is precipitated with isopropanol and cleaned with ethanol. By the end of this

process, there is a visibly pelleted DNA sample that is then preserved in TE and stored at 4ᵒC.

Tissue CollectionCorn plants are field grown at the Agronomy Research Farm. Each plant is given a

unique identifier to tie the plant in the field to the eventual PCR results. A small segment

of leaf tissue was collected and freeze dried in preparation for DNA extraction.

Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (Figure 3) is a repetitive process using a thermocycler that allows the

amplification of a targeted section of DNA. DNA is denatured to allow the attachment of primers

(seen in Figure 2) to the targeted section, where complementary strands of DNA are synthesized

using Taq polymerase, eventually generating millions of copies of the target sequence .

DNA QuantificationDuring DNA Quantification, the nucleic acid concentration of the DNA samples is determined

using a spectrophotometer. This provides the data necessary to appropriately dilute the DNA

samples for use in PCR.

Gel Electrophoresis Gel Electrophoresis is used to determine the alleles in the PCR product of each sample.

During this process, the PCR product is loaded into an agarose gel that has electricity run

through it. The DNA segments move through the gel, creating bands visible under UV light

that indicate the number of base pairs they contain. When observed, samples containing

two bands are determined to be heterozygous while samples containing one band are

determined to be homozygous, allowing for the determination of which plants to continue

breeding for the eventual analysis of a specific allele (Figure 2).

METHODS

• Total plants with DNA isolated and quantified: 632

• Backcross families represented: 81

• Total plants genotyped at locus of interest: 528 or >84%

of total plants

• 76 of the 81 backcross families have been

genotyped

• 50 plants have missing data

• 54 plants have incomplete genotype data or have

not been attempted

• Total primer pairs used: 14

Polymerase chain reaction (PCR)-based genotyping of maize backcross families

B B A H H B B H H A B A H A A H B H H H A H B H

A H B B H B B B A A H H A A B B H H B B A A H H

B73 (A) Mo17 (B)

249 bp>249 bp

insertion

Examples of gel electrophoresis results:

B73 (A) Mo17 (B)

>354 bp354 bp

insertion

B B B B H H B HH HB H B B B B A H B B A A A B

B73 (A) Mo17 (B)

>270 bp270 bp

insertion

RESULTS

Prim

er

pa

ir 1

Prim

er

pa

ir 2

Prim

er

pa

ir 3

DISCUSSION• Plants identified as heterozygous are selected for backcross breeding in this summer’s nursery

as a part of the ongoing process to develop plants containing the target genomic region

• In cases where plants were unsuccessfully genotyped, they will be re-genotyped under

new conditions or with new primer pairs

• Continuing forward, the process of genotyping and backcrossing selected plants will be

repeated across several seasons to achieve the desired analysis families

ACKNOWLEDGMENTSThank you to the YES program and to the Lauter and Yandeau-Nelson lab

groups for this amazing experience. Funding sources for this project include

USDA-ARS Project 5030-21000-060-00D and NSF-IOS Award #1354799.

Backcrossing to one recurrent genotype (B73 or Mo17) generates corn plants that contain a small segment of DNA from

the other genotype at the genomic locus of interest, allowing the specific effects of the DNA segment of interest on lipid

production to be investigated (Figure 1). Several generations of genetic marker-assisted breeding must be performed to

produce materials that segregate for only a small chromosomal segment (Figure 2). The genetic markers used in this

case are PCR amplicon-length polymorphisms resolved by gel electrophoresis (Figure 3 and RESULTS). These data

will guide imminent production of the genetic families needed for biochemical genetic analysis of lipid production.

Figure 2. Inheritance pattern of target section

B73 (A) Mo17 (B)

X =PCR Primer Location

H

H

A

AH = Heterozygous

Figure 3. Polymerase chain reaction

DNA

Sample Dena

tura

tion

Anne

alin

g

DN

A

Synth

esis

Repe

titio

n

Primer

Target DNA

Figure 1. Pictoral representation of backcross breeding: moving Mo17:chr 4S into the B73 genome

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

(B73xMo17)F2 plant with a meiotic cross-over event on the short arm of chromosome 4

Backcross1 (BC1) plant showing ~75% B73 DNA after crossing the F2

recombinant to B73

BC2 plant showing ~87.5% B73 DNA. Compared to the BC1, residual heterozygosity is cut from ~25% to ~12.5%

BC3 plant showing a distal cross-over event on 4S and completion of background conversion