1. Fall 2016 - HORT6033 Molecular Plant Breeding
Instructor: Ainong Shi

2. Fall 2016 - HORT6033 Molecular Plant Breeding
Lecture 2 (08/24/2016)
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. Molecular Plant Breeding
Fall HORT6033
Molecular Plant Breeding
Name
Major
Sumandeep Kaur Bazzer
CSES
Gehendra Bhattarai
CEMB
Yheni Dwiningsih
Amanda Holder
Wade Stiles Hummer
Jamison,Daniel Reed
David Octor Moseley
Joseph Edward Najjar
Akshita Mishra
Bhuvan Pathak
Eliott Emery Pruett
Waltram Ravelombola
Adam David Rice
Xeniya Valeriivna Rudolf
Wei Yang
PLSC
Melinda Yin
HORT
Jamie Lynn Underwood
I. Say Hello
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!

4. Molecular Plant Breeding
Fall 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.
PCR Reaction Youtube Movie
PCR Reaction Youtube Talk
Fig from
Please click links!

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 two tools:
BatchPrimer3
Primer-BLAST
>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. Primer Design Using Primer-BLAST

8. 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]
For example: Shi et al Molecular Markers for Tm-2 Alleles of Tomato Mosaic Virus Resistance in Tomato. American Journal of Plant Sciences 2: [pdf]

9. Primer Design Using BatchPrimer3

10. 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

11. Molecular Plant Breeding
Fall HORT6033
Molecular Plant Breeding
IV. Project
The research project for the Fall Semester, 2016 will be
SSRs and SNPs discovery and validation in cowpea or spinach;
Genetic diversity and population structure in cowpea or spinach;
Association mapping and SNP discovery in cowpea or spinach ; and
QTL mapping for downy mildew resistance in spinach.

12. Molecular Plant Breeding
Fall HORT6033
Molecular Plant Breeding
IV. Homework
Design allele-specific primers for tm-2, Tm-2, and Tm-2a using BatchPrimer3 and Primer-BLAST using the sequences [fasta]
Reading PCR design articles!
For example: Shi et al Molecular Markers for Tm-2 Alleles of Tomato Mosaic Virus Resistance in Tomato. American Journal of Plant Sciences 2:
Click [pdf] to download !

13. SSRLocator for class on Friday
Download ssrlocator and Firebird at
Install Firebird fist and then 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”
Read the manuals at web or download the manuals.
And more …….
Linked