Fall 2016 - HORT6033 Molecular Plant Breeding Instructor: Ainong Shi

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)

Molecular Plant Breeding Fall 2016 - HORT6033 Molecular Plant Breeding Name Email Major Sumandeep Kaur Bazzer skbazzer@uark.edu CSES Gehendra Bhattarai gb005@uark.edu CEMB Yheni Dwiningsih ydwining@uark.edu Amanda Holder alholder@uark.edu Wade Stiles Hummer wshummer@uark.edu Jamison,Daniel Reed djamison@uark.edu David Octor Moseley domosele@uark.edu Joseph Edward Najjar jenajjar@uark.edu Akshita Mishra am068@uark.edu Bhuvan Pathak bppathak@uark.edu Eliott Emery Pruett exp009@uark.edu Waltram Ravelombola wravelom@uark.edu Adam David Rice adamrice@uark.edu Xeniya Valeriivna Rudolf xrudolf@uark.edu Wei Yang wxy008@uark.edu PLSC Melinda Yin mhyin@uark.edu HORT Jamie Lynn Underwood junderwo@uark.edu  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!

Molecular Plant Breeding Fall 2016 - HORT6033 Molecular Plant Breeding II. Estimation Question Discussion (5 min) Click HERE for the Questions! Click Here for Question Answer! http://comp.uark.edu/~ashi/MB/HORT6033_Estimation_question.pdf http://comp.uark.edu/~ashi/MB/goodday/HORT6033_Estimation_question_answer.pdf

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. http://comp.uark.edu/~ashi/MB/pcr.html PCR Reaction Youtube Movie PCR Reaction Youtube Talk Fig from https://www.neb.com/ Please click links!

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 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

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/33330975?report=fasta For example: 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 http://probes.pw.usda.gov/batchprimer3/

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

Molecular Plant Breeding Fall 2016 - 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.

Molecular Plant Breeding Fall 2016 - 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] http://comp.uark.edu/~ashi/MB/example/tomato_Tm2.fasta Reading PCR design articles! For example: 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 Click [pdf] to download !

SSRLocator for class on Friday Download ssrlocator and Firebird at http://cgfufpel.org/ssrlocator_pt.htm. 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