Fall 2014 HORT6033 Molecular plant breeding Instructor: Ainong Shi HORT6033 web site: http://comp.uark.edu/~ashi/MB

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)

I. Say Hello Good morning, everyone! Nice to see you again! Name Major Email Marlovi Andrea Acuna Galindo CSES macunaga@uark.edu Abeer Muhammedali Jasim Al-Nasrawi CEMB amalnasr@email.uark.edu FNU Anuj Kumar axk018@email.uark.edu Jessica L Chitwood HORT jlchitwo@email.uark.edu Marcos Paulo Da Silva mpdasilv@email.uark.edu Mirta Beatriz Dalzotto mbdalzot@uark.edu Terrence James Frett PLSC tjfrett@email.uark.edu Clinton Philip Greub cpgreub@email.uark.edu Ashley Elizabeth Humphreys ENTO aemeyer@email.uark.edu Avjinder Singh Kaler askaler@email.uark.edu Mariola Klepadlo klepadlo@uark.edu Laura Melissa Lara Santisteban lmlarasa@uark.edu Jennifer Anne Lewter jlewter@uark.edu Cindy Massiel Lopez Ramirez cmlopezr@uark.edu Dennis Nicuh Bulusan Lozada dblozada@email.uark.edu Reiofeli Algodon Salas rasalas@email.uark.edu Shilpa Singh shilpa@email.uark.edu Vijay Singh vijay@email.uark.edu Brant Michael Smith PLPA bms010@email.uark.edu Janithri S. Wickramanayake jswickra@uark.edu 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 Email Jianbing Ma jxm044@uark.edu Weiguo Lu 123bean@163.com Haizheng Xiong 390696518@qq.com Jun Qin hbnkydd@163.com Long Yan Dragonyan1979@163.com Maria Nelly Arguello Blanco mnarguellob@gmail.com Liliana Florez-Palacios sandrafp@email.uark.edu

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!

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 https://www.neb.com/) http://comp.uark.edu/~ashi/MB/pcr.html

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 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

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] 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/cgi-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” 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 http://www2.ufpel.edu.br/faem/fitotecnia/fitomelhoramento/faleconosco.html http://www2.ufpel.edu.br/faem/fitotecnia/fitomelhoramento/faleconosco.html

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!

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. 2011. American Journal of Plant Sciences 2:180-189 [pdf]