1. Fall 2014 HORT6033 Molecular plant breeding
Instructor: Ainong Shi
HORT6033 web site:

2. Molecular Plant Breeding
Fall 2014 HORT6033
Molecular Plant Breeding
Lecture 2 (08/25/2014)
Say Hello Each Other (5 min)
Estimation Question Discussion (5 min)
PCR and Primer Design (30 min)
Research Project and Homework (8 min)
Questions (2 min)

3. I. Say Hello Good morning, everyone! Nice to see you again!
Name
Major
Marlovi Andrea Acuna Galindo
CSES
Abeer Muhammedali Jasim Al-Nasrawi
CEMB
FNU Anuj Kumar
Jessica L Chitwood
HORT
Marcos Paulo Da Silva
Mirta Beatriz Dalzotto
Terrence James Frett
PLSC
Clinton Philip Greub
Ashley Elizabeth Humphreys
ENTO
Avjinder Singh Kaler
Mariola Klepadlo
Laura Melissa Lara Santisteban
Jennifer Anne Lewter
Cindy Massiel Lopez Ramirez
Dennis Nicuh Bulusan Lozada
Reiofeli Algodon Salas
Shilpa Singh
Vijay Singh
Brant Michael Smith
PLPA
Janithri S. Wickramanayake
Good morning, everyone!
Nice to see you again!
I am Ainong Shi, as you know, the teacher for this class. I speak “Chinese English” in the class,
and apologize for my strong accent. I am the vegetable breeder in the Department of Horticulture.
My hobby is “watching Chinese Kung Fu Movies”.
Please introduce yourself!
Name
Jianbing Ma
Weiguo Lu
Haizheng Xiong
Jun Qin
Long Yan
Maria Nelly Arguello Blanco
Liliana Florez-Palacios

4. Molecular Plant Breeding
Fall 2014 HORT6033
Molecular Plant Breeding
II. Estimation Question Discussion (5 min)
Click HERE for the Questions!
Click Here for Question Answer!

5. III. PCR and Primer Design
Developed in 1983 by Kary Mulli,
who won the Noble Prize for the PCR technology.
PCR (polymerase chain reaction) is a technology used to amplify a single copy of DNA segment to millions of copies. And then you can see by eyes or machines.
The PCR reaction consists of Denaturation, Annealing, and Extension.
Fig from

6. III. PCR and Primer Design
2. PCR Primer Design
Dozens of PCR primer design programs and tools are available for free to use from the Internet. We only discuss three tools:
BatchPrimer3
Primer-BLAST
SSRLocator
>FG938745_UCRVU09_UCR 707 TTTTTTTTTTTTTTTTTACCCAGAAACAGCTTAGAGCGTGAAAGCAAACACTAATGAACAAACCTTGAAAAGGCTTTATATATATACACAGTACACTTAGCAAAAGCACGCCCATACCTGCGTTTTTACATATTTACATCTAACCGACCATAACCATAAACACTGGACAAACTAAGACTCAGCTGTTAAACCACACAAAGCTACCACCATTTCAACCGCTTAACTCATGAACTCATACATATATATATATATATATATATATATATATATATATATATACACACACATATATATGCACAAAAAGGTAAACAAAAATGATACAGCATGACCAAAAAAGGCAGAGGAAAGCAAACTATATTTTCATTCTTTTAGTGATCATGGAGAGTCATTTCTCACACATCGAAAGGAGAAGAAGAGCTTGGTCTGTTCTGGCGAGTCTTCTCGAACGCATCCGATGTCGACCTTCTTCGAAACCGGAAGGACTCTCTAGCTCCTGAAAACGGAGACACCGGAGGAGTAGAACCGGCGGGTGAAGCCGGCGCCGATCCACTCTGACTCTGATATCCGGGTGGCTTCACGATCATGATACTACGTGTAACTCTCATCGCGTCTTCCGGCGAATCCTCACCGTATGACTTCA CGCTTCCGCCGTCTGATTCCTTGCCAGAG
.fasta format
Example 1: One cowpea EST multiple alleles
Example 2: cowpea ESTs with SNPs mapped to the cowpea genetic maps
Example 3: Tomato Mosaic Virus resistance gene Tm-2 multiple alleles

7. Tomato Mosaic Virus resistance gene tm2,Tm-2, and Tm-2a [fasta]
Primer Design using Primer-BLAST
Design PCR primers using Primer3 and search the forward and reverse primers in all available in GenBank.
For example:
Tomato Mosaic Virus resistance gene tm2,Tm-2, and Tm-2a [fasta]
Shi et al Molecular Markers for Tm-2 Alleles of Tomato Mosaic Virus Resistance in Tomato American Journal of Plant Sciences 2: [pdf]

8. Primer Design using BatchPrimer3
BatchPrimer3 is a tool for designing various primers including for SSRs
It can be used for SSR discovery as well
Tool website:
Web tool:
Examples
cowpea_EST_1_0693.fasta
Pick up part of the EST sequences from below file, cowpea_SSR_SNP_geneticMap_marker.fasta

9. Primer Design using SSRLocator
Install SSRLocator in C: as showing C:\SSRLocatorI
Copy your data file with fasta format (.fasta) into the SSRLocatorI folder such as “cowpea_EST_1_0693.fasta”
Open SSRLocatorI.exe
Format file to SSRLocator Standard in the “Create File” button. Save a new file called “cowpea_EST_1_0693format.fasta”
And more …….
Linked

10. Molecular Plant Breeding
Fall 2014 HORT6033
Molecular Plant Breeding
Lecture 2 (08/27/2014)
IV. Project and Homework
The research project for the Fall Semester, 2014 will be
SSRs and SNPs discovery and validation in chicory or other crops;
Genetic diversity in cowpea or other crops;
Trait : markers association analysis in cowpea;
Association mapping in spinach or other crops; and
QTL mapping for downy mildew resistance in spinach or others.
Each Student will work one more project.
[pdf]
You can work your own project!

11. Molecular Plant Breeding
Fall 2014 HORT6033
Molecular Plant Breeding
Lecture 2 (08/27/2014)
Homework
Discuss SSRLocator installation
Discuss how to download cowpea ESTs from GenBank
Design allele-specific primers for tm-2, Tm-2, and Tm-2a
Read the article Shi et al American Journal of Plant Sciences 2:
[pdf]