Application of next generation sequencing of a begomovirus-

resistant inbred to design a KASPar® assay for SNP detection of

the Ty1-Ty3 region

Menda, N.1, S. Strickler1, D.M. Dunham1, D.P. Maxwell2, L. Mejia2, G.B. Martin1, and L.A.

Mueller1

1) Boyce Thompson Institute for Plant Research,

Cornell University, Ithaca, NY, USA 2) Semillas Tropicales, S.A., Guatemala

Luis Mejia San Carlos University (Retired) Semillas Tropicales Guatemala

Guatemala, Central America TYLCV and several bipartite begomoviruses

FAVI 9 - (I902 x S)

• Resistant • Hybrid • 150 g fruit • Determinant • V, F1, F2

Favi Vidavski

Hebrew University of Jerusalem

Gh13 inbred selected from Favi 9 (h902 x S inbred)

Gh13 early Oct. 2012 Commercial Hybrid

Opportunity to use WGS to search for introgressions (TBRT meeting, Ithaca, NY 2011)

Nov.2012

SGN Staff: “Re-sequencing assembly” of Gh13 WGS using Heinz

1706 as the reference sequence • 20X coverage, Illumina sequencing

• http://bio-animations.blogspot.com/2011/02/dna-sequencing-illumina.html

• Assembly: reference sequence and de novo

• GBrowse available (SGN site) • SNP density plots (10-kb window)

Mi

Ty3 ↑

↑

↑ Ty1 TG97

Gh13 compared with Heinz1706

SNP plot

# nt’s in gaps/10 kb

# SNPs/10 kb

How does the SNP plot data from whole genome sequencing of Gh13 compare with data from the SolCap

Illumina SNP chip for Chromosome 6?

Solcap Illumina Chip – UC-Davis Allen van Dynze

SolCap code Mbp Gh13-R HUJ-S Poly SGN solcap_snp_sl_44591 30.373 BB BB M

solcap_snp_sl_44607 30.6 BB BB M 30.600

solcap_snp_sl_100297 30.623 BB BB M

solcap_snp_sl_100298 30.624 AA BB P

solcap_snp_sl_100549 30.925 BB AA P

solcap_snp_sl_44625 30.926 BB AA P

solcap_snp_sl_44633 31.039 BB BB M

solcap_snp_sl_44650 31.222 AA BB P

solcap_snp_sl_17476 31.305 BB AA P

solcap_snp_sl_68976 33.706 BB AA P

solcap_snp_sl_100365 33.775 AA BB P

solcap_snp_sl_69004 33.783 NC BB M

solcap_snp_sl_100142 33.809 AA BB P

solcap_snp_sl_55858 33.973 BB AA P

solcap_snp_sl_55874 34.12 AA AA M 34.213

solcap_snp_sl_55880 34.266 AA AA M

solcap_snp_sl_55902 34.396 BB BB M

Verlaan, M.G., D. Szinay, S.F. Hutton, H. de Jong, R. Kormelink, R.G.F. Visser, J.W. Scott, Y. Bai. Chromososomal rearrangements between tomato and Solanum chilense hamper mapping and breeding of the TYLCV resistance gene Ty-1. Plant J. (2011) 68:1093-1103.

Source of Ty1: two commercial hybrids; Ty1 from S. chilense LA1969 Source of Ty3: Scott-Hutton program, S. chilense LA2779

“Disease tests on progenies of informative recombinants with TYLCV mapped Ty-1 to the long arm between markers MSC05732-4 and MSc05732-14, an interval overlapping with the reported Ty-3 region, which led to the indication that Ty-1 and Ty-3 may be allelic.” Verlaan et al., 2011

Verlaan et al., 2011

↑ ↑

Ty1-Ty3 30.6 to 30.9 Mbp

Ty3, SCAR Marker (31.5, P6-25)

WGS – SNP density plot ch06: 30.6 to 34.213 Mbp

30.6

34.2

30.6

30.9

Gbrowse (SGN) Verlaan et al.

Marker: P6-25F2/P6-25R5

(Ji, Jensen, Melgar, Maxwell, Sept. 07)

Ty3b, 660 bp = S. chilense LA1969 (100%)

ty3 Ty3 Ty3a Ty3b Ty3b (Chromosome 6, 31.498 Mbp)

AS-PCR, detection of a SNP (G/A) KASP, from LGC Genomics

A

G

A = Resistant genotype G = Susceptible genotype

Common primer reverse sense

Forward sense, with FAM or VIC

Fluorescent reading of 96, 384, 1536 well plates

(http://www.lgcgenomics.com/how-does-kasp-work)

Q

Q

Ag Biotech uses KASP technology for SNP detection of tomato traits

Assured Excellence in Plant Genetic Analysis

SL2.40ch06:30,600,000..30,900,000 Ty1-Ty3 region, Verlaan et al.

Example of Strategy Pick target gene or region Design PCR primers Sequence fragments Align sequences, pick SNP Design KASP primers

An Example of KASPar Primer design

PCR fragment sequence alignment

inbred locus forward primer sequence SNP 1-30 nt reverse primer

Heinz ty 24-30 nt, 40-50% GC's T 24-30 nt, 40-50%

GC's

Purple Russian ty same sequence T same sequence

Gh13-WGS Ty3 same sequence G same sequence

Gh13-PCR Ty3 same sequence G same sequence

Gc9 (LA2779) Ty1-Ty3 same sequence G same sequence Inbred-ST1 (LA2779) Ty1-Ty3 same sequence G same sequence Gc171 (LA1932) Ty3a same sequence G same sequence

LA1969 Ty3b same sequence G same sequence

KASPar Assay on known genotypes

T G

G = Resistant genotype (RED) T = Susceptible genotype (BLUE) Het = (Green)

Common primer reverse sense

Forward sense, with FAM or VIC

SNP170 – Ag Biotech

Q

Q

Genotype CALL

Germplasm Mi TG97 (Ty1) P6-25 (Ty3) SNP170 Purple Russian S S S S HUJ-VF S S S S Heinz 1706 S S S S M82 S S S S Motelle R S S S Anahu R S S S Rodeo R S S S Celebrity R S S S Amelia R S S S Plum Crimson R S S S Gh13 S S Ty3/Ty3 R Gc9 S Ty1/Ty1 Ty3/Ty3 R Gc171 S S Ty3a/Ty3a R Inbred-Ty1 (LA1969) S Ty1/Ty1 Ty3b/Ty3b R Llanero** R S Ty3a/ty3 Het Marwa** ? Ty1/ty1 Ty3b/ty3 Het Tritiet** R Ty1/ty1 Ty3b/ty3 Het

SNP170 – Ag BioTech

384 well plate, with data from one 96 well plate Each well contained leaves from a different plant from F3 families for Sem. Trop.

Advantages of SNP170

• Does not require agarose gels (P6-25), so is much faster

• Closer to the “true” resistance determinant than SCAR marker P6-25; should give fewer false positives

• No false positives with Mi resistant germplasm • Positive calls for Ty1, Ty3, Ty3a, Ty3b • Adapted to large scale screening using 384 or

1536 well plates or other platforms

Disadvantages of SNP170

• Does not distinguish between Ty1, Ty3, Ty3a, or Ty3b

• Cost of equipment for reading fluorescent dyes, much more than agarose gels

Special Thanks • East-West Seeds for hosting TBRT • Citizens of the USA

– NSF grant to SGN for sequencing, staff at SGN – SolCap grant for SNP genotyping – SolCAP SNP

platform – USDA funds for preparation of DNA – CDR/MERC grants for P6-25 and TG97 analysis

• Ag Biotech – Testing KASPar primers and verification of SNP170

• Semillas Tropicales – Supplying Gh13 and some other lines for testing