1. Fall 2016 HORT6033 Molecular Plant Breeding
Instructor: Ainong Shi
HORT6033 web site:

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

3. 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. (

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

5. SNP Discovery Tool
There are dozens of tools for SNP discovery. We will talk below two:
BioEdit
Download:
Manual:
DNASTAR Lasergene Genomics Suite
Download free TRY IT version : products-dnastar-lasergene-genomics.aspx and all use manuals can also be downloaded or viewed from the web site.

6. SNP Genotyping Single SNP genotyping assay:
Taqman – Life Technologies
KASP genotyping - LGC Genomics
Multiple SNP genotyping:
SNP chip – Illumina
Multiplex – Sequenom
SNP genotyping by next generation sequencing
Genotyping by sequencing (GBS) (
Restriction site associated DNA sequencing (RADseq) (

7. 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

8. SNP Discovery Using BioEdit
Download and Install BioEdit
Example data: Sw-5 of gene of tomato spotted wilt virus resistance
Run BioEdit and Find SNPs
Example – SNP discovery in Tomato
resistance-2009aps.ppt

9. 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

10. 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

11. Reading
Varshney et al Next-generation sequencing technologies and their implications for crop genetics and breeding. Trends in Biotechnology 27:522–530 (Linked)
Liu et al An improved allele-specific PCR primer design method for SNP marker analysis and its application. Plant Methods 8: 34 ( pdf)
Gaur et al High-Throughput SNP Discovery and Genotyping for Constructing a Saturated Linkage Map of Chickpea (Cicer arietinum L.). DNA Res 19 (5): (
Shi et al Identification of molecular markers for Sw-5 gene of tomato spotted wilt virus resistance. American Journal of Biotechnology and Molecular Sciences (1): [PDF]

12. 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