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

2. Fall 2016 - HORT6033 Molecular Plant Breeding
Lecture 3 SSR (08/26/2016)
Class PCR and Primer Design
SSR
SSRLocator
Homework
Questions

3. SSR Marker and Tools After PCR, the DNA sample with Allele #1, #2, #3
SSR is repeating sequences of 2-5 (most of them) base pairs of DNA such as (AT)n, (CTC)n, (GAGT)n, (CTCGA)n
SSR is called Microsatellite
SSR usually is co-dominant
SSR can be used:
Fingerprinting
Genetic diversity
Genetic mapping
Association mapping
Marker-assisted selection
Genome-wide selection etc.
Left Figure:
Allele #1: (CA)11 [SSR motif (CA) repeat 11 times]
Allele #2: (CA)13
Allele #3: (CA)16
After PCR, the DNA sample with Allele #1, #2, #3
showing different size DNA fragment and the
different combination of allele #1, #2, and #3 with different pattern with co-dominant.
(image from

4. Example for SSR (AT) from BatchPrimer3

5. SSRLocator
SSR discovery for large DNA sequences, and also for primer design and PCR simulation (Maia et al Int J Plant Genomics :412696) [PDF]
Tools:
Example: cowpea_EST_1_0693.fasta (linked)

6. Download and Install SSRLocator
Download SSRLocator and Firebird at Both FireBird (database for store SSR data) and SSRLocator are needed for run the software.
The installation manuals can be viewed at
Farebird: .
SSRLocator:
Download manuals at
- Install FareBird DataBase - (pdf) - Install SSRLocator - (pdf)
The user manual can be viewed at

7. SSR Discovery 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

8. SSRLocator - Output
The SSR motif (AT) creates various repeats from 12 to 20 among the five cowpea samples (below Loci in the Table). Primer pairs are designed and expected PCR size are estimated ranged 211 to 381 bp. The polymorphism can be used as marker – SSR marker !
Contig
Loci
Start_SSR1
End_SSR1
FG938745_UCRVU09_UCR_707
(AT)20
239
278
FG908248_UCRVU08_CCNS5607_IT97K-461-4
(AT)13
204
229
FG875445_UCRVU07_CCNP10713_UCR_779
(AT)12
205
228
FG850994_UCRVU05_CCNN17188_IT84S-2049
(AT)19
200
237
FG815463_UCRVU04_CCNI2988_524B
(AT)15
216
245
Contig
Forward TM
Reverse
Product
Size
FG938745
AACAGCTTAGAGCGTGAAAG
55.1
TTTCGATGTGTGAGAAATGA
381
FG908248
CAGCTGTTAAACCACACAAA
54.9
213
FG875445
211
FG850994
224
FG815463
216

9. SSR Discovery Using SSRLocator
Online
Linked
Sequence data

10. Molecular Plant Breeding
Fall HORT6033
Molecular Plant Breeding
Homework
Download cowpea ESTs, run SSRLocator, and find SSRs.
2. Read SSR locator manual and SSR articles.

11. Homework 1 for week 1

12. Homework 1 for week 1 FALL 2016 HORT6033
Genetic Techniques in Plant Breeding (Molecular Plant Breeding)
Homework 1 – week one (8/22 to 8/25/2016)
Read the course syllabus at
Read the class presentations (download from
Install SSRLocator! Please read the guide and manuals! Please check more info at the course web page at
Examples will be found at our web site:
Example 1:
Example 2 :
For example 1 EST data, please run it for SSR discovery and primer design using BatchPrimer3 at !
Please do: copy example 1 data and paste them to the Textbox in the tool interface. Click “Pick Primers” button! That’s it and you will find the SSRs and the SSR primer pairs!
Run example 1 and 2 in SSRLocator
Extra:
Please read the article:
Maia et al Int J Plant Genomics. 2008:412696) at
Wei et al Characterization of the sesame (Sesamum indicum L.) global transcriptome using Illumina paired-end sequencing and development of EST-SSR markers. BMC Genomics 2011, 12:451 (
Run Example 2 using SSRLocator
Download all cowpea (Vigna unguiculata) ESTs from GenBank (we will discuss how to download million sequences from GenBank and save them to a local file.)
Download and install BioEdit at
Download and install MEGA 6 at
Run example 1 in BioEdit and MEGA 6
Run all cowpea EST data in SSRLocator!
Homework due:
Right now, homework to me at anytime!
Homework 1 should be due Sunday night (August 29). Each student should hand on the results and the summary of SSR discovery and also the primers (Monday class).
Computer software and tool imitations, running, and exercise are counted as credit. Please have a list for all tools which you install, and you can run examples on it. The list should be ed to me before the final exam. We will give you a test whether you can use the software or tools. The course requires you can run >= 10 tools. You can gain 5% for running more tools.
Please also write out what articles you read during the semester and a simple summary for each article which you have read should be hang on.