Fall 2016 HORT6033 Molecular Plant Breeding Instructor: Ainong Shi HORT6033 web site: http://comp.uark.edu/~ashi/MB

Molecular Plant Breeding Fall 2016 HORT6033 Molecular Plant Breeding Lecture 4 (08/29/2016) SNP SNP discovery by PCR Example Homework Question

Single Nucleotide Polymorphism (SNP) A single nucleotide (A, T, C or G) mutation Use: genetic and genome mapping, Association studies, Genetic diversity analysis, and Tagging important genes Approach: SNP discovery from PCR SNP discovery from High throughput - Next generation sequencing (NGS) – RNA-Seq, RAD-Seq, GBS, WGS, WGR, etc. (http://learn.genetics.utah.edu/content/pharma/snips/)

Single Nucleotide Polymorphism (SNP) An example for Mi Motella/LA2823 Mi AAGTAGACGAGGTTAGTAAAAT Mogeor/LA3471 Mi AAGTAGACGAGGTTAGTAAAAT NY07-464 mi AAGTAGACGACGTTAGTAAAAT LA3130 mi AAGTAGACGACGTTAGTAAAAT Motella Mogeor G G NY07-464 LA3130 C C

SNP Discovery Tool There are dozens of tools for SNP discovery. We will talk below two: BioEdit Download: http://www.mbio.ncsu.edu/bioedit/page2.html Manual: http://www.mbio.ncsu.edu/BioEdit/BioDoc.pdf DNASTAR Lasergene Genomics Suite Download free TRY IT version : http://www.dnastar.com/t- products-dnastar-lasergene-genomics.aspx and all use manuals can also be downloaded or viewed from the web site.

SNP Genotyping Single SNP genotyping assay: Taqman – Life Technologies http://www.vetmed.ucdavis.edu/vme/taqmanservice/pdfs/SNP_Genotyping.pdf KASP genotyping - LGC Genomics http://www.lgcgenomics.com/genotyping/kasp-genotyping-chemistry/how-does-kasp-work/ Multiple SNP genotyping: SNP chip – Illumina Multiplex – Sequenom SNP genotyping by next generation sequencing Genotyping by sequencing (GBS) (http://cbsu.tc.cornell.edu/lab/doc/GBS_Method_Overview1.pdf) Restriction site associated DNA sequencing (RADseq) (http://bfg.oxfordjournals.org/content/9/5-6/416.full.pdf+html)

SNP Discovery by PCR Primer Design PCR PCR Product Sequencing AAGCTTGGTGCAATGACATTATACAATCAACAAAGAATAGAAAGGAGATAAAAAGCACTATCCAAATCCC --------------------------------------------------------------------- ---------------------------------------------------------------------- TCCATTTTCCTTTTTTTGATTATTCCAGCTACCAAGACAACCACTAGGGGCAGTCCTTTGCATTTTTCTG CTACTGCTTGACTCACATCTTGTAGTTCAAGCGGGCAATCTTCCTTTTGAAACACTTTTTTCTGCAACAA TTGGCAACTCTCTTCTGTTGTGAGGAATGGAAGAGAATAAGGATCAGTATGGTACTTGACTTGCTTACCC ACTTCTTCAAGTCGAGTTGTTACGACTATTCTGCTTCTAATTCCAACATCTGGAAAAGAAAGCCTTAAGT CATCCCATACCATACAATCCCACATATCATCCAATACAATGAGATATCTCTTTCCCATTAATTTTCTCCT CAACATGTCGGCAAGAACATCAACCGTAGCTCCATTGTCGTTGAAACCTGTAACTTGACTGAAAATATCT TGTAATAACTCTATCCGGTTATACGTTTGAGAAATGATGCACCATGCTCGAACATCAAAGCGAGAAACAA TAATGTCATTATTGTACAACTTTCTAGCAATTGTCGTTTTCCCTTGTCCTCCCATGCCTACAATTGGGAC AACGTCTAGCTCATTTGTACCTCTAGTCAGATAATCAATTAGTTCTTCTGCTTGTTTCTCAAAACCCACT ACCTCCTCATCAGTTACAAGATTGCTATGTCGATCCGGCAGATGTTTTAATGGAGCAGCCACATAGTGAG TTGGCTTAAAATAAGTTTTTTTTAACTTAAAATATGTTATTTTGAGTATTGACAAACAACTAAATAAGTC AAAAATCAGCTTTTAAGTCAGTTTGACCACGCTCTCTTAT Sw-5: AY007366 Tool: Primer-BLAST Primer Design Sw5-f1 AACCACTAGGGGCAGTCCTT Sw5-r1 CTCACTATGTGGCTGCTCCA (TGGAGCAGCCACATAGTGAG) PCR PCR Product Sequencing Multiple Sequence Alignment Accession Gene sw5-snp Stevens Sw-5b 31392 A LA3667 331 LA3433 sw-5b G LA2823 SNP Discovery SNP Validation SNP Genotyping

SNP Discovery Using BioEdit Download and Install BioEdit http://www.mbio.ncsu.edu/bioedit/bioedit.html Example data: Sw-5 of gene of tomato spotted wilt virus resistance http://comp.uark.edu/~ashi/MB/example/Sw-5/sw5.fasta Run BioEdit and Find SNPs Example – SNP discovery in Tomato http://comp.uark.edu/~ashi/MB/lecture/Shi_et_al_SNP-assay-tomato-disease- resistance-2009aps.ppt

Molecular Plant Breeding Fall 2015 HORT6033 Molecular Plant Breeding Lecture 5 (08/29/2016) Homework Download Tm-2 gene sequences from NCBI web site (GenBank), run BioEdit to find SNP markers for Tm-2 locus of tomato mosaic virus resistanc Download Sw-5 gene sequences from GenBank and find SNP markers for Sw-5 alleles of spotted wilt virus resistance

Multiple Sequence Alignment SNP Markers for Tm-2 locus in Tomato AF536199_tm-2 TGATCGACGTAGCCTCATTCAACTTCCTTCTGGTGTTTGGGAGTCTAAACATTTGAGACATCTTTGTTAT TGGAGGGGAATATTTGTGGAAAACTGCCAAATAGTATTGTCAAGCTCACACGTCTAGAGACCATAGACAT ----------------------------------------------------------------- AGAGATTATGGACAAGCATGTAACAGTTGCTTTTCTATAAGCTCATTTTACCCAAACATTTACTCATTGC CCTGTGCCAAAGGCGCTAAAGGTTCTGAAGCTCAGGTTTTTCAGTGATCCGAGTGAGCAAATAAACTTGT GATCAATTTAAGAAAACTGGGTATACTGGGAGTGTCCAATTCAACCGTTAAGATATTATCAACATGTCGC ATCCTAACAATCTACAAACCTTGATGTGGATACCTGATAAATTTTTTGAACCGAGGTTGTTGCACCGATT ACATTACCCAAATTAAGAAAACTTAAAATGGTCATCTGCAAGTATAATGAAGAAAAGATGGCTCTCTCGG CCCTCCAAATATTATCAAGCTTACTCTTGTCTGCTTTATGGTAGACAGTTGTCTACTGGCAGTGCTTAAG CATCCTATCCAAAAATTGTTAAGTTGCATTTGAATGTTGACAGAACAATAGCCTTGAACTCTGAAGCATT ------------------------------------------------------------____------ GCGAGGCAAATGGTTATAGCTTTCCGCAACTTGAAGTTTTGCATATTCATAGCCCGAATGGGTTGTCTGA AF536201_Tm2a TGATCGACGTAGCCTCATTCAACCTCCTTCTGGTGTTTGGGAGTCTAAACATTTGAGACATCTTTGTTAT TGGAGGGGAATATTTGTGGAAAACTGCCAAATAGTATTGTCAAGCTCACACGTCTAGAGACCATAGACAT ----------------------------------------------------------------- AGAGATTATGGACAAGCATGTAACAGTTGCTTTTCTATAAGCTCATTTTACCCAAATATTTACTCATTGC CCTGTGCTTAAGGCGCTGGAGGTTCTGAAGCTCAGTTTTTCCAGTGACCCGAGTGAACAAATAAAGTTGT GATCAATTTAAGAAAACTGGGTATACTGGGAGTGTCCAATTCTACCGTTAAGATGTTATCAATATTTAGC ATCCTAACAATCTACAAACCTTGATGTGGATACCTGATAAATTTTTTGAACCGAGGTTGTTGCACCGATT ACATTTCCCAAATTAAGAAAACTTAAAATGTTCATCTGCAAGTATAATGAAGAAAAGATGGATCTCTCGG TCCTCCAAATCTCATCAAGCTTACTCTAGCCTACTTTAGTGTAGACCGTTATATACTGGCAGTACTTAAG CATCGTATCCACATATTGCTAAGTTGCATTTGAATGTTAACAGAACAATGGCCTTGAACTCTCAATCATT -----------------------------------------------------___-------------- GCGAGGCAAATGGTTATAGCTTTCCGCAACTTGAAGTTTTGCATATTCATAGCCCGAATGGGTTGTCTGA AF536200_Tm2 TGATCGACGTAGCCTCATTCAACCTCCTTCTGGTGTTTGGGAGTCTAAACATTTGAGACATCTTTGTTAT TGGAGGGGAATATTTGTGGAAAACTGCCAAATAGTATTGTCAAGCTCACACGTCTAGAGACCATAGACAT ----------------------------------------------------------------- AGAGATTATGGACAAGCATGTAACAGTTGCTTTTCTATAAGCTCATTTTACCCAAATATTTACTCATTGC CCTGTGCTCAAGGCGCTGGAGGTTCTGAAGCTCAGTTTTTCCAGTGACCCGAGTGAACAAATAAAGTTGT GATCAATTTAAGAAAACTGGGTATACTGGGAGTGTCCAATTCTACCGTTAAGATGTTATCAATATTTAGC ATCCTAACAATCTACAAACCTTGATGTGGATACCTGATAAATTTTTTGAACCGAGGTTGTTGCACCGATT ACATTTCCCAAATTAAGAAAACTTAAAATGTTCATCTGCAAGTATAATGAAGAAAAGATGGATCTCTCGG TCCTCCAAATCTCATCAAGCTTACTCTAGCCAACTTTACGGTAGACCGTTATATACTGGCAGTACTTAAG CATCGTATCCACATATTGCTAAGTTGCATTTGAATGTTAACAGAACAATGGCCTTGAACTCTCAATCATT -----------------------------------------____------------------------- GCGAGGCAAATGGTTATAGCTTTCCGCAACTTGAAGTTTTGCATATTCATAGCCCGAATGGGTTGTCTGA Tool: Primer-BLAST Primer Design Tm2RS-f3 TGGAGGGGAATATTTGTGGA Tm2RS-r3 ACTTCAGACAACCCATTCGG (CCGAATGGGTTGTCTGAAGT) PCR PCR Product Sequencing Multiple Sequence Alignment SNP Discovery CAPS Marker Identification SNP Genotyping SNP Genotyping

Reading Varshney et al. 2009. Next-generation sequencing technologies and their implications for crop genetics and breeding. Trends in Biotechnology 27:522–530 (Linked) Liu et al. 2012. An improved allele-specific PCR primer design method for SNP marker analysis and its application. Plant Methods 8: 34 (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3495711/pdf/1746-4811- 8-34.pdf) Gaur et al. 2012. High-Throughput SNP Discovery and Genotyping for Constructing a Saturated Linkage Map of Chickpea (Cicer arietinum L.). DNA Res 19 (5): 357-373 (http://dnaresearch.oxfordjournals.org/content/19/5/357.full) Shi et al. 2011. Identification of molecular markers for Sw-5 gene of tomato spotted wilt virus resistance. American Journal of Biotechnology and Molecular Sciences (1): 8-16. [PDF] http://scihub.org/AJBMS/PDF/2011/1/AJBMS-1-1-8-16.pdf

Development of SNP Multiplex Assays for Disease Resistance Genes in Tomato For example Ainong Shi and Richard Vierling Indiana Crop Improvement Association (Purdue University) Richard Grazzini GardenGenetics LLC http://comp.uark.edu/~ashi/Ainong_UARK/presentation/presentation/2009aps_tomato.pdf