Transcript PCR and primer design

FALL 2014
HORT6033
MOLECULAR
PLANT BREEDING
Instructor: Ainong Shi
HORT6033 web site: http://comp.uark.edu/~ashi/MB
Fall 2014 HORT6033
Molecular Plant Breeding
Lecture 2 (08/25/2014)
I. Say Hello Each Other (5 min)
II. Estimation Question Discussion (5 min)
III. PCR and Primer Design (30 min)
IV. Research Project and Homework (8 min)
V.Questions (2 min)
I. Say Hello
Name
Major
Email
Marlovi Andrea Acuna Galindo
CSES
[email protected]
Abeer Muhammedali Jasim AlNasrawi
CEMB
[email protected]
FNU Anuj Kumar
CSES
[email protected]
Jessica L Chitwood
HORT
[email protected]
Marcos Paulo Da Silva
CSES
[email protected]
Mirta Beatriz Dalzotto
CSES
[email protected]
Terrence James Frett
PLSC
[email protected]
Clinton Philip Greub
CEMB
[email protected]
Ashley Elizabeth Humphreys
ENTO
[email protected]
Avjinder Singh Kaler
CSES
[email protected]
Mariola Klepadlo
CSES
[email protected]
Laura Melissa Lara Santisteban
CSES
[email protected]
Jennifer Anne Lewter
PLSC
[email protected]
Cindy Massiel Lopez Ramirez
CSES
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
Dennis Nicuh Bulusan Lozada
CEMB
[email protected]
Reiofeli Algodon Salas
CSES
[email protected]
Shilpa Singh
CEMB
[email protected]
Vijay Singh
CSES
[email protected]
[email protected]
Brant Michael Smith
PLPA
[email protected]
[email protected]
Janithri S. Wickramanayake
ENTO
[email protected]
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
Email
Fall 2014 HORT6033
Molecular Plant Breeding
II. Estimation Question Discussion (5 min)
Click HERE for the Questions!
Click Here for Question Answer!
III. PCR and Primer Design
1. PCR
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.
http://comp.uark.edu/~ashi/MB/pcr.html
Fig from https://www.neb.com/)
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
.fasta format
>FG938745_UCRVU09_UCR 707
TTTTTTTTTTTTTTTTTACCCAGAAACAGCTTAGAGCGTGAAAGCAAACACTAATGAACAAACCTTGAAAA
GGCTTTATATATATACACAGTACACTTAGCAAAAGCACGCCCATACCTGCGTTTTTACATATTTACATCT
AACCGACCATAACCATAAACACTGGACAAACTAAGACTCAGCTGTTAAACCACACAAAGCTACC
ACCATTTCAACCGCTTAACTCATGAACTCATACATATATATATATATATATATATATATATATATATATATATAC
ACACACATATATATGCACAAAAAGGTAAACAAAAATGATACAGCATGACCAAAAAAGGCAGAGG
AAAGCAAACTATATTTTCATTCTTTTAGTGATCATGGAGAGTCATTTCTCACACATCGAAAGGAGAAGA
AGAGCTTGGTCTGTTCTGGCGAGTCTTCTCGAACGCATCCGATGTCGACCTTCTTCGAAACCGGAA
GGACTCTCTAGCTCCTGAAAACGGAGACACCGGAGGAGTAGAACCGGCGGGTGAAGCCGGC
GCCGATCCACTCTGACTCTGATATCCGGGTGGCTTCACGATCATGATACTACGTGTAACTCTCATCG
CGTCTTCCGGCGAATCCTCACCGTATGACTTCA CGCTTCCGCCGTCTGATTCCTTGCCAGAG
Example 1: One cowpea EST multiple alleles
http://comp.uark.edu/~ashi/MB/example/cowpea_EST_1_0693.fasta
Example 2: cowpea ESTs with SNPs mapped to the cowpea genetic maps
http://comp.uark.edu/~ashi/MB/example/cowpea_SSR_SNP_geneticMap_marker.fasta
Example 3: Tomato Mosaic Virus resistance gene Tm-2 multiple alleles
http://comp.uark.edu/~ashi/MB/example/tomato_Tm2.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]
http://www.ncbi.nlm.nih.gov/nuccore
Shi et al. 2011. Molecular Markers for Tm-2 Alleles of Tomato Mosaic Virus
Resistance in Tomato American Journal of Plant Sciences 2:180-189 [pdf]
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:
http://batchprimer3.bioinformatics.ucdavis.edu
• Web tool:
http://batchprimer3.bioinformatics.ucdavis.edu/c
gi-bin/batchprimer3/batchprimer3.cgi
• Examples
cowpea_EST_1_0693.fasta
Pick up part of the EST sequences from below file,
cowpea_SSR_SNP_geneticMap_marker.fasta
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” Linked
 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 …….
http://www2.ufpel.ed
u.br/faem/fitotecnia/
http://www2.ufpel.edu.br/f
fitomelhoramento/fal
econosco.html
aem/fitotecnia/fitomelhora
mento/faleconosco.html
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
1.
SSRs and SNPs discovery and validation in chicory or other crops;
2.
Genetic diversity in cowpea or other crops;
3.
Trait : markers association analysis in cowpea;
4.
Association mapping in spinach or other crops; and
5.
QTL mapping for downy mildew resistance in spinach or others.
Each Student will work one more project.
You can work your own project!
[pdf]
Fall 2014 HORT6033
Molecular Plant Breeding
Lecture 2 (08/27/2014)
Homework
1.
Discuss SSRLocator installation
2.
Discuss how to download cowpea ESTs
from GenBank
3.
Design allele-specific primers for tm-2,
Tm-2, and Tm-2a
4.
Read the article Shi et al. 2011. American
Journal of Plant Sciences 2:180-189 [pdf]