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FALL 2014 HORT6033 MOLECULAR PLANT BREEDING Instructor: Ainong Shi HORT6033 web site:

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Presentation on theme: "FALL 2014 HORT6033 MOLECULAR PLANT BREEDING Instructor: Ainong Shi HORT6033 web site:"— Presentation transcript:

1 FALL 2014 HORT6033 MOLECULAR PLANT BREEDING Instructor: Ainong Shi HORT6033 web site: http://comp.uark.edu/~ashi/MBhttp://comp.uark.edu/~ashi/MB

2 Fall 2014 HORT6033 Molecular Plant Breeding Lecture 2 (08/25/2014) I.Say Hello Each Other (5 min) II. Estimation Question Discussion (5 min) III. PCR and Primer Design (30 min) IV. Research Project and Homework (8 min) V.Questions (2 min)

3 I. Say Hello NameMajorEmail Marlovi Andrea Acuna GalindoCSESmacunaga@uark.edu Abeer Muhammedali Jasim Al- Nasrawi CEMBamalnasr@email.uark.edu FNU Anuj KumarCSESaxk018@email.uark.edu Jessica L ChitwoodHORTjlchitwo@email.uark.edu Marcos Paulo Da SilvaCSESmpdasilv@email.uark.edu Mirta Beatriz DalzottoCSESmbdalzot@uark.edu Terrence James FrettPLSCtjfrett@email.uark.edu Clinton Philip GreubCEMBcpgreub@email.uark.edu Ashley Elizabeth HumphreysENTOaemeyer@email.uark.edu Avjinder Singh KalerCSESaskaler@email.uark.edu Mariola KlepadloCSESklepadlo@uark.edu Laura Melissa Lara SantistebanCSESlmlarasa@uark.edu Jennifer Anne LewterPLSCjlewter@uark.edu Cindy Massiel Lopez RamirezCSEScmlopezr@uark.edu Dennis Nicuh Bulusan LozadaCEMBdblozada@email.uark.edu Reiofeli Algodon SalasCSESrasalas@email.uark.edu Shilpa SinghCEMBshilpa@email.uark.edu Vijay SinghCSESvijay@email.uark.edu Brant Michael SmithPLPAbms010@email.uark.edu Janithri S. WickramanayakeENTOjswickra@uark.edu NameEmail 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 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!HERE Click Here for Question Answer!Here

5 III. PCR and Primer Design 1. PCR Fig from https://www.neb.com/) 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

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 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.fasta format >FG938745_UCRVU09_UCR 707 TTTTTTTTTTTTTTTTTACCCAGAAACAGCTTAGAGCGTGAAAGCAAACACTAATGAACAAACCTTGAAAA GGCTTTATATATATACACAGTACACTTAGCAAAAGCACGCCCATACCTGCGTTTTTACATATTTACATCT AACCGACCATAACCATAAACACTGGACAAACTAAGACTCAGCTGTTAAACCACACAAAGCTACC ACCATTTCAACCGCTTAACTCATGAACTCATACATATATATATATATATATATATATATATATATATATATATAC ACACACATATATATGCACAAAAAGGTAAACAAAAATGATACAGCATGACCAAAAAAGGCAGAGG AAAGCAAACTATATTTTCATTCTTTTAGTGATCATGGAGAGTCATTTCTCACACATCGAAAGGAGAAGA AGAGCTTGGTCTGTTCTGGCGAGTCTTCTCGAACGCATCCGATGTCGACCTTCTTCGAAACCGGAA GGACTCTCTAGCTCCTGAAAACGGAGACACCGGAGGAGTAGAACCGGCGGGTGAAGCCGGC GCCGATCCACTCTGACTCTGATATCCGGGTGGCTTCACGATCATGATACTACGTGTAACTCTCATCG CGTCTTCCGGCGAATCCTCACCGTATGACTTCA CGCTTCCGCCGTCTGATTCCTTGCCAGAG

7 Primer Design using Primer-BLAST Design PCR primers using Primer3 and search the forward and reverse primers in all available in GenBank. 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]pdf http://www.ncbi.nlm.nih.gov/nuccore For example: Tomato Mosaic Virus resistance gene tm2,Tm-2, and Tm-2a [fasta]fasta

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 http://batchprimer3.bioinformatics.ucdavis.edu Web tool: http://batchprimer3.bioinformatics.ucdavis.edu/c gi-bin/batchprimer3/batchprimer3.cgi http://batchprimer3.bioinformatics.ucdavis.edu/c gi-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 cowpea_SSR_SNP_geneticMap_marker.fasta

9 Primer Design using SSRLocator http://www2.ufpel.ed u.br/faem/fitotecnia/ fitomelhoramento/fal econosco.html  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 ……. http://www2.ufpel.edu.br/f aem/fitotecnia/fitomelhora mento/faleconosco.html Linked

10 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 1. SSRs and SNPs discovery and validation in chicory or other crops; 2. Genetic diversity in cowpea or other crops; 3. Trait : markers association analysis in cowpea; 4. Association mapping in spinach or other crops; and 5. QTL mapping for downy mildew resistance in spinach or others. [pdf]pdf You can work your own project! Each Student will work one more project.

11 Fall 2014 HORT6033 Molecular Plant Breeding Lecture 2 (08/27/2014) Homework 1. Discuss SSRLocator installation 2. Discuss how to download cowpea ESTs from GenBank 3. Design allele-specific primers for tm-2, Tm-2, and Tm-2a 4. Read the article Shi et al. 2011. American Journal of Plant Sciences 2:180-189 [pdf]pdf


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