Molecular Markers for Tm-2 Alleles of Tomato Mosaic Virus Resistance in Tomato

doi:10.4236/ajps.2011.22020

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American Journal of Plant Sciences, 2011, 2, 180-189

doi:10.4236/ajps.2011.22020 Published Online June 2011 (http://www.SciRP.org/journal/ajps)

Molecular Markers for Tm-2 Alleles of Tomato

Mosaic Virus Resistance in Tomato

Ainong Shi1,2*, Richard Vierling2,6, Richa rd Gra zzini3, Pengyin Chen4, Homer Caton1, Dilip Panthee5

1Syngenta Seeds, Slater, USA; 2Indiana Crop Improvement Association and Department of Agronomy, Purdue University, West

Lafayette, USA; 3GardenGenetics LLC, Bellefonte, USA; 4Department of Crop, Soil, and Environmental Sciences, University of

Arkansas, Fayetteville, USA; 5Department of Horticultural Science, North Carolina State University, Mountain Horticultural Crops

Research and Extension Center, Raleigh, USA; National Corn Growers Association, Cepi Drive Chesterfield, USA; 6National Corn

Growers Association, Chesterfield, USA.

Email: *ainong.shi@syngenta.com, ainong_shi@hotmail.com

Received January 23rd, 2011; revised March 26th, 2011; accepted March 31st, 2011.

ABSTRACT

Tomato mosaic virus (To MV) is one of the most infectious virus diseases in tomato (Solanum lycopersicum L). The

practical and effective method o f controllin g this disease is through gene tic control by u sing major resistance genes. So

far, three genes Tm-1, Tm-2 and Tm-22 conferring resistance to ToMV have been reported and utilized in toma to culti-

var development. Marker assisted selection (MAS) has become very important and useful tool in selection of ToMV re-

sistant tomato lines or hybrids. The objective of this research was to identify allele-specific PCR-bas ed, cleaved ampli-

fied polymorphic sequence (CAPS), and allele-derived single nucleotide polymorphism (SNP) markers for Tm-2 loci.

Four allele-specific PCR-based markers were iden tified: one for Tm-2, one for Tm-22, and two for th e susceptib le allele

tm-2. Three allele-derived CAPS markers were identified, which can identify and distinguish three alleles, tm-2, Tm-2

and Tm-22 in tomato germplasm. Three SNP markers were developed specific for Tm-2 locus. These markers will pro-

vide breeders with a tool in selection of Tm-2 and Tm-22 resistance genes i n tomato breeding pr ogr a m.

Keywords: Cleaved Amplified PolymorphicSequence,Marker-Assisted Selection, Single Nucleotide Polymorphism,

Solanum Lycopersicum, Tomato, Tomato Mosaic Virus (ToMV), Tm -2

1. Introduction

Tomato mosaic virus (ToMV) is one of the most infec-

tious virus diseases in tomato (Solanum lycopersicum L.

formerly, Lycopersicon esculentum Mill). The practical

and effective method of controlling this disease is

through introducing major resistance gene(s). So far,

three genes, Tm-1, Tm-2 and Tm-22 of ToMV resistance

have been reported and used in tomato cultivar develop-

ment [1-5]. The Tm-1 gene was introgressed into S. ly-

copersicum from the wild tomato species S. habrochaites

and mapped to chromosome 5 and it conferred resistance

to ToMV strains 0 and 2 [1,4,6,7]. The Tm-1 gene has

been cloned and the sequences were stored in GenBank

with five accessions DJ344478 to DJ344481, and

DJ344505. The other two genes Tm-2 and Tm-22 were

introgressed from S. peruvianum and were located closely

to the centromere of chromosome 9 and are considered to

be allelic and Tm-2 conferred resistance to ToMV strains

0 and 1, whereas allele Tm-22 conferred resistance to

ToMV strains 0, 1 and 2 [2,3,5,8-10].

Molecular markers have been widely used in genetic

mapping and marker-assisted selection (MAS) for dis-

ease resistance in tomato [6]. For Tm-2 genes of ToMV

resistance, Ohmori et al. [11] reported 13 random ampli-

fied polymorphic DNA (RAPD) markers linked to the

Tm-2 locus. From the 13 RAPD markers, Motoyoshi et al.

[11] found two markers, OPE16 (900) and OPN31 (1000),

nearest to the Tm-2 locus. Six out of the 13 RAPD mark-

ers distributed within 0.7 cM on chromosome 9 were

cloned and sequenced to be converted into sequence

characterized amplified region (SCAR) markers [11].

Sobir et al. [12] and Dax et al. [13] identified co-dominant

SCAR markers tightly linked to Tm-22. Three alleles,

Tm-2, Tm-22, and tm-2 at Tm - 2 locus have been cloned

and sequenced (GenBank Accessions: AF536199,

AF536200, and AF536201) and three cleaved amplified

polymorphic sequences (CAPS) markers were developed

to distinguish the three alleles in tomato [14]. Based on

the two CAPS markers reported by Lanfemeijer et al.

Molecular Markers for Tm-2 Alleles of Tomato Mosaic Virus Resistance in Tomato181

[14], Arens et al. [15] developed a co-dominant assay

combining tetra primers designed from the SNP regions

of the two CAPS markers using a method called

ARMS-PCR SNP and this Tetra-primer ARMS-PCR

assays can amplify PCR products with different DNA

fragment sizes from tomato lines carrying Tm-2 or Tm-22,

and susceptible allele tm-2.

Single nucleotide polymorphism (SNP) is becoming to

be the most useful as molecular marker in genome map-

ping, association studies, diversity analysis, and tagging

genes for economically important traits in crop plants

because of their abundance and automated highthroughput

genotyping [16-18]. SNPs have been discovered and

verified in tomato [19-22] and successfully used in se-

lecting resistance to bacterial speck and bacterial spot in

tomato [22,23]. The objective of this research was to

identify Tm-2 allele-specific PCR-based markers, CAPS

markers, and SNP markers for MAS in tomato breeding.

2. Materials and Methods

2.1. Plant Materials

Twenty-three tomato genotypes including released or

commercial cultivars and accessions were used in this

research (Table 1). Seeds of 15 tomato accessions (LA

series) were obtained from the C.M. Rick Tomato Genetics

Resource Center, Dept. of Plant Sciences, University of

California, Davis, CA 95616 (http://tgrc.ucdavis.edu), six

cultivars were purchased from commercial sources, and

two lines NY07-461 and NY07-464 were obtained from

Cornell University; refined selections from a cross be-

tween “Brandywine” and “Rose de Berne” (received as

NY07-461 (Brandyrose #1) and NY07-464 (Brandyrose

#2)). Among the 23 genotypes, three cultivars, Royal

Red (LA2088), CLN2264F (LA4285), and LA3433 are

homozygous for Tm-2 (Tm-2/Tm-2); Mogeor (LA3471),

Sophya, and VFNT Cherry (LA1221) are homozygous

for Tm-22 (Tm-22/Tm-22); Swt Cluster, Bush Celebrity,

and Golden Girl are heterozygous for Tm-22 (Tm-22/tm-2);

and the other 14 genotypes contained the susceptible

allele tm-2 (Table 1).

2.2. DNA Extraction, PCR Amplification and

DNA Sequencing

Genomic DNA was extracted from fresh leaves of

greenhouse-grown plants using the CTAB (Cetyl-

trimethylammonium Bromide) method [24,25]. The

gene- specific primers for Tm-2, Tm-22, or tm-2 at Tm-2

locus were designed from the sequences of the GenBank

accessions AF536199, AF536200, and AF536201 using

the primer design tool—Primer-BLAST (http://www.ncbi.

nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=Bl

astHome). AF536199 is the accession in GenBank that

contains the sequence of the tm-2 susceptible allele;

AF536200 is the sequence Tm-2 resistance allele; and

AF536201 is the sequence of Tm-22 resistance allele. The

size of the AF536199 sequence of the allele tm-2 is 2820

bp; AF536200 is 2819 bp; and AF536201 is 9837 bp [3].

Primers Tm2RS-f2, Tm2RS-r2, Tm2RS-f3, and Tm2RS-

r3 were designed for the three sequences of AF536199,

AF536200, and AF536201; Tm2S-f1, Tm2S-r1, Tm2S-f2,

and Tm2S-r2 for AF536199 only; Tm2R-f1c and Tm2R-

r4 for both AF536200 and AF536201; Tm2R-r3 for

AF536200 only; and Tm2aR-r3 for AF536201 only (Ta-

ble 2).

PCR amplification was performed in an eppendorf

thermal cycler (Eppendorf, Westbury, NY) following

standard PCR procedures with minor modifications.

Briefly, each 50 µl PCR reaction mixture consisted of

29.8 µl sterilized ddH2O, 10 µl 5x Mango Taq reaction

buffer (Bioline, London, UK), 3 µl MgCl2 (25 mM), 1.5

µl dNTP (2.5 mM each), 1.5 µl each primer (5 µM), 0.2

µl Mango Taq DNA polymerase (5 U/µl) (Bioline, Lon-

don, UK), and 2.5 µl template DNA (30 ng/µl). For the

primer pairs, Tm2RS-f2/r2 and Tm2RS-f3/r3, PCR pro-

cedure consisted of an initial denaturation step of 94˚C

for 4 min followed by 36 to 38 cycles of 15 to 30 second

at 94˚C for denaturation, 15 to 30 s at 51 to 56˚C for an-

nealing, and 40 to 75 s at 72˚C for extension depending

on primer pairs with a final extension step at 72˚C for 5

min (Table 3). The PCR fragments were separated by gel

electrophoresis with 1.5% agarose gel in 0.5 X TAE

buffer, stained with ethidium bromide, and visualized

with UV light.

The PCR fragments were sequenced in the Purdue

Genomics Core Facility, Purdue University, West Lafay-

ette, IN 47907. Before sequencing of PCR products, PCR

products were purified using QIA quick PCR Purification

Kit (Qiagen Inc, Valencia, CA) following the manufac-

turer’s instructions of the protocol. The sequences ampli-

fied from the two primer pairs Tm2RS-f2/-r2 and Tm2RS-

f3/-r3 in tomato were submitted to GenBank using a

DNA sequence submission and update tool—Sequin

(http://www.ncbi.nlm.nih.gov/Sequin/).

2.3. SNP Identification and Genotyping

SNP discovery was postulated from available Tm-2 se-

quences of AF536199, AF536200, and AF536201. The

multiple sequence alignment showed that 68 SNPs and

one InDel were observed among the three sequences.

One of the approaches to SNP validation is through

re-sequencing of the same gene among various genotypes.

Two Tm-2 gene-specific primer pairs Tm-2RS-f2/r2 and

Tm-2RS-f3/r3 were designed from the sequences of

AF536199, AF536200, and AF536201 using Primer-

LAST (Table 2) and used to run PCR among tomato B

Molecular Markers for Tm-2 Alleles of Tomato Mosaic Virus Resistance in Tomato

182

Table 1. Allele-specific PCR-based, allele-derived CAPS, and allele-derived SNP markers for Tm-2 loci in 23 tomato geno-

types.

Allele-specific PCRa Allele-derived CAPS

SNP genotyping

by Sequenom b

Cultivar/

accession

Genotype

at Tm-2

locus Tm2S-f1/

Tn2S-r1 Tm2S-f2/

Tm2S-r2

Tm2R-f1

Tm2R-r3

Tm2R-f1c/

Tm2aR-r3

Tm2R-fi

c/Tm2-r

Tm2rs-

f3/r3 PshAIHpaI BsiHKAI

Tm2-

snp1 Tm2-

snp46 Tm2-

snp53

NY07-461 tm-2/tm-2 + + − − − 703 bp538 bp