FALL 2014HORT6033

MOLECULAR PLANT BREEDINGInstructor: Ainong Shi

Fall 2014 HORT6033Molecular Plant Breeding

Lecture 1 (08/25/2014)

I. Class Overview (5 min)II.Class Estimation Questions and

Answers (15 min)III.SSR Marker and Tools (25 min)IV. Homework (3 min)V.Questions (2 min)

Fall 2014 HORT6033

Molecular Plant BreedingInstructor: Dr. Ainong Shi

Phone: 479-575-2670 (o), 479-879-0159 (c)Email: ashi@uark.eduOffice: PTSC 321Office Hours: Please email to Ainong Shi at ashi@uark.edu to set up an appointment. But feel free to call or stop by. Class Hours: MWF 9:40 – 10:30 am, August 25 – December 19, 2014Classroom: UAF campus AGRI 0301ALab Location: PTSC 322, open: 7 days a week Lab Assistant: Dr. Jianbing Ma (jxm044@uark.edu)Guest Instructor: Dr. John R. Clark, Dr. Pengyin Chen, Dr. Richard Esten Mason, Dr. Andy Pereira, Dr. Subodh Srivastava, and Dr. Weiguo Lu ………..

Student InfoName Major Email

Marlovi Andrea Acuna Galindo CSES macunaga@uark.eduAbeer Muhammedali Jasim Al-Nasrawi CEMB amalnasr@email.uark.eduFNU Anuj Kumar CSES axk018@email.uark.eduJessica L Chitwood HORT jlchitwo@email.uark.eduMarcos Paulo Da Silva CSES mpdasilv@email.uark.eduMirta Beatriz Dalzotto CSES mbdalzot@uark.eduTerrence James Frett PLSC tjfrett@email.uark.eduClinton Philip Greub CEMB cpgreub@email.uark.eduAshley Elizabeth Humphreys ENTO aemeyer@email.uark.eduAvjinder Singh Kaler CSES askaler@email.uark.eduMariola Klepadlo CSES klepadlo@uark.eduLaura Melissa Lara Santisteban CSES lmlarasa@uark.eduJennifer Anne Lewter PLSC jlewter@uark.eduCindy Massiel Lopez Ramirez CSES cmlopezr@uark.eduDennis Nicuh Bulusan Lozada CEMB dblozada@email.uark.eduReiofeli Algodon Salas CSES rasalas@email.uark.eduShilpa Singh CEMB shilpa@email.uark.eduVijay Singh CSES vijay@email.uark.eduBrant Michael Smith PLPA bms010@email.uark.eduJanithri S. Wickramanayake ENTO jswickra@uark.edu

Name EmailJianbing Ma jxm044@uark.edu

Weiguo Lu 123bean@163.com

Haizheng Xiong 390696518@qq.com

Jun Qin hbnkydd@163.com

Long Yan Dragonyan1979@163.comMaria Nelly Arguello Blanco

mnarguellob@gmail.com

Liliana Florez-Palacios sandrafp@email.uark.edu

Register

Fall 2014 HORT6033

Molecular Plant Breeding

Project

Lecture Lab

Tools, Homework, Reading, and Research

Fall 2014 HORT6033Syllabus of Molecular Plant Breeding

I. Course DescriptionII. Course Objective and OutcomeIII. GradingIV. Class ScheduleV. Software and Tools Download and InstallationVI. Quiz and Examination (30%)VII. Assignment (homework, computer tools, and reading) (30% +

5% bonus)VIII. Lab, Project, Report, Presentation, and Article Reading (30%

+ 10% bonus)IX. Reading Materials

Download Syllabus at http://comp.uark.edu/~ashi/MB/HORT6033_syllabus.pdf

Course Web: http://comp.uark.edu/~ashi/MB

Fall 2014 HORT6033Molecular Plant Breeding

Lecture 1 (08/25/2014)

II. Class Estimation Questions and Answers

This is just an estimation to test which level each student hasbefore the class, in order to divide students into groups. Download the questions at

1. Quiz

2. Quiz discussion

http://comp.uark.edu/~ashi/MB/HORT6033_Estimation_question.pdf

http://comp.uark.edu/~ashi/MB/goodday/HORT6033_estimation_question_answer_08252014.pdf

III. SSR Marker and Tools

(image from http://www.cdfd.org.in/)

• SSR is repeating sequences of 2-5 (most of them) base pairs of DNAsuch as (AT)n, (CTC)n, (GAGT)n, (CTCGA)n

• SSR is called Microsatellites

• 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, #3showing different size DNA fragment and the different combination of allele #1, #2, and #3 With different pattern with co-dominant.

Example for SSR (AT)

1. SSRLocator SSR discovery for large DNA sequences, and also

for primer design and PCR simulation (Maia et al. 2008. Int J Plant Genomics. 2008:412696)[PDF]

Tools: from Plant Genomics and Breeding Center - Federal University of Pelotas – Brazil ( ufpel.edu.br ).

Example: cowpea_EST_1_0693.fasta (linked)

http://www2.ufpel.edu.br/faem/fitotecnia/fitomelhoramento/faleconosco.html

1. SSRLocator - running

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 Standart in the “Create File” button. Save a new file called “cowpea_EST_1_0693format.fasta”

And more …….

1. SSRLocator - Output

Contig LociStart_SSR

1 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

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 FowardTMFoward

ReverseTMReverse

ProductSize

FG938745 AACAGCTTAGAGCGTGAAAG 55.1 TTTCGATGTGTGAGAAATGA 55.1 381

FG908248 CAGCTGTTAAACCACACAAA 54.9 TTTCGATGTGTGAGAAATGA 55.1 213

FG875445 CAGCTGTTAAACCACACAAA 54.9 TTTCGATGTGTGAGAAATGA 55.1 211

FG850994 CAGCTGTTAAACCACACAAA 54.9 TTTCGATGTGTGAGAAATGA 55.1 224

FG815463 CAGCTGTTAAACCACACAAA 54.9 TTTCGATGTGTGAGAAATGA 55.1 216

2. MEGA and BioEdit

MEGA 6 and BioEdit can be used to view SSR motifs among DNA samples (above from MEGA 6 and the bottom from BioEdit)

3. BatchPrimer3

• BatchPrimer3 is the tool for primer design including

• Primers 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/cgi-bin/batchprimer3/batchprimer3.cgi

• An example cowpea_EST_1_0693.fasta”.

Fall 2014 HORT6033Molecular Plant Breeding

Lecture 1 (08/25/2014)

IV. Homework1. Download cowpea ESTs from GenBank

2. Download and Install software SSRLocator

3. Read SSRlocator manual and SSR articles

4. Download MEGA 6, BioEdit and manuals

Fall 2014 HORT6033Molecular Plant Breeding

Lecture 1 (08/25/2014)

V. Homework1. Download cowpea ESTs from

GenBank

V. Homework

2. Download and Install software SSRLocator

Download the tools from Plant Genomics and Breeding Center - Federal University of Pelotas – Brazil ( ufpel.edu.br ). The tools for Windows Seven 32 bits should still work for Windows 64. You should download FireBird (database for store SSR data) and SSRLocator. The In the web page, you can see the link for Install Instruction and User Guide. You should also download or view the instruction for FireBird and SSRLocator, and also for User Guide!

V. Homework

3. Read SSR locator manual and SSR articles

Manual download!

SSR article:

THANKS!

Dear Students: Please read and download homework and quiz athttp://comp.uark.edu/~ashi/MB/molecularBreeding_homework.html !