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Fall 2014 HORT6033 Molecular plant breeding

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). Say Hello E ach Other (5 min) Estimation Question Discussion (5 min)

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Fall 2014 HORT6033 Molecular plant breeding

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  1. Fall 2014HORT6033Molecular plant breeding Instructor: Ainong Shi HORT6033 web site: http://comp.uark.edu/~ashi/MB

  2. 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)

  3. 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!

  4. Fall 2014 HORT6033 Molecular Plant Breeding • II. Estimation Question Discussion (5 min) Click HERE for the Questions! Click Here for Question Answer!

  5. III. PCR and Primer Design 1. PCR Developed in 1983 by KaryMulli, 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

  6. 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

  7. 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]

  8. 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

  9. 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

  10. Fall 2014 HORT6033 Molecular Plant Breeding Lecture 2 (08/27/2014) 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. • IV. Project and Homework Each Student will work one more project. [pdf] You can work your own project!

  11. Fall 2014 HORT6033 Molecular Plant Breeding Lecture 2 (08/27/2014) • 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 • Homework [pdf]

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