Download THE SCREENING OF SEVERAL MOLDAVIAN TOMATO

Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi
s. II a. Biologie vegetală, 2012, 58, 1: 5-10
http://www.bio.uaic.ro/publicatii/anale_vegetala/anale_veg_index.html
ISSN: 1223-6578, E-ISSN: 2247-2711
THE SCREENING OF SEVERAL MOLDAVIAN TOMATO CULTIVARS FOR
IDENTIFICATION OF MI-NEMATODE RESISTANCE GENE
Maria DUCA1*, Angela PORT1, Antonina CROITORU1, Nadejda VRANCEAN2, Isgouhi
KALOSHIAN3
Abstract: Twenty two tomato cultivars grown in the Republic of Moldova were screened for resistance to
root-knot nematode by molecular marker analysis and nematode bioassay. DNA based test and nematode
bioassay were used to determine the presence of the root-knot nematode resistance gene Mi in these
tomato cultivars. PCR product digestion with Taq I enzyme was performed to distinguish the homozygous
and the heterozygous state of a particular allele at the Mi locus. One tomato line (348-III-2) was identified
as homozygous resistant for the Mi gene, one variety (Djina) and two lines (35-IX-1, 351-IV-1) as
heterozygous. The rest of the tomato genotypes tested were homozygous for the susceptible allele.
Nematode bioassays were in agreement with the molecular marker analysis. This screening represents the
first study of resistance to root-knot nematodes of tomatoes cultivated in the Republic of Moldova.
Keywords: Root-knot nematode - GI- Meloidogyne - Solanum lycopersicon - Mi
Introduction
Tomato (Solanum lycopersicon L.) (Peralta and Spooner, 2001) is the second most
consumed vegetable after potato (FAOSTAT, 2010), widely grown around the world, and
constitutes a major agricultural industry especially for agricultural countries such as the
Republic of Moldova. The tomato crop is seriously affected by over 200 diseases caused by
pathogenic fungi, bacteria, viruses and nematodes (Abad Z. G. and Abad J. A., 1997; Koch
et al. 1992; Lukyanenko, 1991; Moriones and Navas-Castillo, 2000); therefore a major goal
of modern tomato breeding programs is the development of cultivars with improved disease
resistance.
The tomato resistance gene Mi encodes a protein with nucleotide-binding and
leucine-rich repeat domains and is located near the centromere of the short arm of
chromosome 6 (Mehlenbacher, 1995; Milligan et al., 1998). It was introgressed into
cultivated tomato from its wild relative Solanum peruvianum, using traditional breeding
and embryo rescue (Smith, 1944). The gene confers resistance against three major
Meloidogyne species that infect tomato, M. arenaria, M. incognita and M. javanica
(Holliday, 1989; Martinez de Ilarduya al., 2001; Nombela et al., 2003). Root-knot
nematodes are endoparasites considered to be the most damaging pests of tomato
worldwide (Trudgill and Blok, 2001). Disease symptoms caused by root-knot nematode on
susceptible host plants include damaged root system and the formation of root galls,
wilting, chlorosis, and stunted growth resulting in poor yield (Abad et al., 2003). Mi
mediated resistance is characterized by a rapid (within 12 hours of infection), localized
necrosis or hypersensitive response near the infecting head of the juvenile that limits its
1* University of the Academy of Science of Moldova, Chisinau, Republic of Moldova. [email protected]
(corresponding author)
2 Scientifical-Practical Institute of Phytotechny, Republic of Moldova
3 University of California, Riverside, California, 92521-0124, USA
Duca, M. et al., 2012/ An. Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 58, 1: 5-10
feeding and establishment of a feeding site known as giant cells (Ho et al., 1992; Hwang et
al., 2000). Breeding root knot nematode resistant tomato varieties constitutes an
economically feasible option for nematode management (Cook, 2000). Although nematode
infection sometimes occurs on resistant tomato cultivars, the pest generally fails to develop
and reproduce to high levels on these resistant tomato genotypes grown under greenhouse
or field conditions (Terrell et al., 1983).
Marker assisted selection represent a powerful tool to overcome some limitations of
traditional breeding methods and is successfully adopted by tomato breeding programs.
Marker assisted selection for disease resistance offer the additional advantage of permitting
selection for resistance in the absence of the pathogen(s) (Kelly, 1995; Mehlenbacher,
1995). Among crop species, tomato is very rich in the number of available molecular
markers. Therefore, it is possible to identify and develop an efficient DNA PCR marker that
is tightly linked to the trait of interest.
Rex-1 is a co-dominant PCR–based marker that has been shown to be tightly linked
to the Mi locus and is frequently used as a diagnostic marker to differentiate root-knot
nematode resistant and susceptible tomato cultivars. (Williamson et al., 1994). The
objective of this study was to screen several tomato cultivars grown in Moldova, for
resistance to root-knot nematode using molecular marker assays with perspective to use
them in the development of new nematode resistance hybrid cultivars.
Materials and methods
Plant material. In this study eleven tomato varieties were used: Fakel, Djina,
Missouri, Shakira, Onix, Zagadca, Peto-86, Laguna, Nadejda, Novicioc, Leana. In
addition, two hybrids Dacia and Lira, and nine tomato lines: 528, 13, 8, 346-I-1, 347-IX-1,
348-III-2, 349I-2, 35-IX-1, 351-IV-1 kindly provided by the Scientifical-Practical Institute
of Phytotechy and the Institute of Genetics (Republic of Moldova). Two tomato lines were
used as controls. Cultivar VFNT (Mi/Mi) that is homozygous for the nematode resistant
allele Mi and cultivar UC82B (mi/mi), homozygous for the susceptible allele mi.
The plants were grown in plastic pots of 500 ml volume filled with a mix of steam
sterilized sand and soil (9:1). Every two weeks plants were watered with a mix of organic
and mineral fertilizer of Biohumus Raduga trademark (1 litter solution contains: NH4NO310g; K2O-10g; P2O5-10g; huminic acids-2 g).
Nematode isolation and inoculation. Root-knot nematode infected tomato plants,
grown in a greenhouse where nematode susceptible tomatoes are grown for several years,
were used as the source of the nematode inoculum. Tomato roots were cut to pieces and
blended in a 0.5% NaOCl solution to extract eggs (Hussey and Barker, 1973). The solution
was poured directly through a 200-mesh (75 µm) sieve nested on a 1 l beaker, and then
eggs were collected on a 500-mesh (26 µm) sieve. Eggs were allowed to hatch using a
modified Bearmann funnel (Martinez de Ilarduya al., 2001).
Two plants per genotype were infected at the stage of three true leaves. For the
nematode infection, 1 ml of inoculum was added at the base of each plant in small holes.
Three months after infection, the tomato plants were carefully removed from the pots, and
the root systems washed free of soil. Then, the roots were stained with a 0.05%
erioglaucine (Sigma, Saint Louis, MO, USA), egg masses and galls counted, and analyzed
microscopically (light microscope МБС-9, Russia).
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Duca, M. et al., 2012/ An. Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 58, 1: 5-10
The degree of root knot nematode infection was evaluated by the gall index (GI)
using a scale of 0-5 as follows: 0 - no galls or egg masses on the plant root; 1- 1 to 2 galls
or egg masses; 2 - 3 to 10 galls or egg masses; 3 - from 11 to 30 galls or egg masses; 4 - 31
to 100 galls or egg masses, 5 - more than 100 galls or egg masses per root system.
Analysis of tomato DNA for the REX-1 marker by PCR and digestion with
TaqI. Genomic DNA was isolated from leaves using CTAB method (Doyle J. J. and Doyle
J. L., 1987). DNA quality and yield was evaluated by 0.8% agarose gel electrophoresis and
UV absorbance (A260/A280) using PG instruments UV.VIS NC 60 spectrophotometer (PG
instruments, Great Britain).
PCR amplification was carried out with the following reaction mix: 50 ng of
genomic DNA template, 5 pmol of primers: REX-F1 (5’-TCGGAGCCTTGGTCTGAATT3’) and REX-R2 (5’-GCCAGAGATGATTCGTGAGA-3’) (Williamson et al., 1994); 200
µM dNTP, 2.5 mM MgCl2, 1x PCR buffer provided by the manufacturer, 1.25 units Go Taq
DNA polymerase (Promega, Madison, WI, USA) in a total volume of 25 µl. The PCR
reaction was performed in an automated thermal cycler (CG1-96, Corbett, Austria)
programmed: 940C for 3 min, followed by 35 cycles: 94 °C for 1 min, 55° C for 2 min, 72
°C for 2 min and a final extension cycle at 720C for 5 min.
Amplified products (7 µL) were digested overnight at 65°C using 5 units of TaqI
(Sigma, USA) per reaction (10 µL). Digested products were resolved on a 1.5% agarose gel
and 50 bp DNA ladder (Fermentas, USA) was used to estimate the fragment size.
Results and discussion
Root-knot nematode resistance bioassay. The nematode bioassay revealed typical
disease symptoms caused by root-knot nematodes characterized by the presence of small
galls, 1 to 10 mm in size, on most infected tomato roots (Fig. 1A, B and C). Scoring the
root systems, using the galling index, revealed different levels of susceptibility among the
tomato genotypes tested. Since the root systems of the tomato plants were not large, the
nematode infection rate was low on the susceptible control as well as on the susceptible
genotypes (Fig. 1D). The evaluation of the degree of root galling showed that eleven
varieties and eight lines had GI between 1 and 4 displaying galls with different sizes. Peto86 was scored as the most susceptible genotype. Five genotypes varieties Shakira, Peto-86,
Laguna and line 349-I-2 as well as the susceptible control UC82-B had the highest GI
scores between 3 and 4 (Fig. 1D). The hybrid plants Dacia and Lira had GI of 2 and 1,
respectively, most galls being small in size, <1mm. The tomato genotypes 348-III-2, 35-IX1, as well as the resistant control VFNT showed no root galls.
The susceptible plants displayed also some symptoms of wilting and chlorosis. Since
the vascular alterations caused by the formation of the giant cells restrict water and
nutrients uptake, and interfere with the translocation of minerals and photosynthates in the
host, most likely the plants manifested a reduced capacity for nutrient uptake with a general
reduction in vigor typical symptoms observed on root-knot nematode infested hosts.
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Duca, M. et al., 2012/ An. Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 58, 1: 5-10
A
A
D
B
4
3.
5
3
C
GI
2.
5
2
1.
5
1
0.
5
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20
21 22 23 24
Figure 1. Morpho-physiological evaluation of root gall formation. A. root of a susceptible tomato
(variety Peto-86); B. group of galls; C. magnified view shows the presence of egg masses,
indicating successful reproduction of nematodes; D. root-knot nematode resistance bioassay.
1-Fakel; 2-Djina; 3-UC82-B (negative control); 4-VFNT (positive control); 5-Missouri; 6-348-III-2; 7-Shakira; 8-Onix; 9-Zagadca;
10-Peto-86; 11-Laguna; 12-Nadejda; 13-Novicioc; 14-Leana; 15-346-I-1; 16-35-IX-1; 17-347-IX-1; 18-351-IV-1;19-349-I-2; 20Dacia; 21-528; 22-13; 23-Lira; 24-8.
Molecular screening of tomato genotypes for the presence of the Mi gene. DNA
based test with the PCR marker Rex-1 and post-PCR digestion with the restriction enzyme
Taq I was performed to identify the presence or absence and the zygosity of a particular
allele at the Mi locus.
A single amplified product of 750 base pair (bp) was obtained with all samples using
PCR and the Rex-1 primers (data not shown). Digestion of the PCR products with the Taq I
restriction enzyme results in two fragments of 570 bp and 160 pb, if Mi allele is present in
the homozygous condition (Mi/Mi) as it is shown in the control VFNT tomato sample (Fig.
2 lane 4). If the Mi allele is in the homozygous susceptible condition, then the Rex-1 PCR
product doesn’t contain a Taq I cleavage site and is not restricted as it is in the susceptible
control cultivar UC82B sample (Fig. 2 lane 3).
The molecular test of studied eleven varieties, nine lines and two F1 hybrids showed
that the Mi allele is present in the heterozygous state (Mi/mi) in one variety (Djina) and two
lines (35-IX-1, 351-IV-1) because when resolved electrophoretically these samples yielded
three fragments of 750, 570 and 160 bp in length (Fig. 2 lanes 2, 16, and 18). One tomato
line (348-III-2) displayed the homozygous Mi resistant banding pattern (Fig. 2 lane 6). The
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Duca, M. et al., 2012/ An. Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 58, 1: 5-10
rest of tomato samples, including the hybrid lines Dacia and Lira, displayed homozygous
mi susceptible banding pattern (Fig. 2).
M 1 2
3
4 5
S R/S S R
6 7 8
S R S S
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
S S S S S
S S R/S S R/S S S S
S S
S
Figure 2. Gel electrophoresis of the digested amplicons. M - molecular marker; lane 1 - Fakel; lane
2 - Djina; lane 3 - UC82B (susceptible control); lane 4 - VFNT (resistant control); lane 5 Missouri; lane 6 - 348-III-2; lane 7 - Shakira; lane 8 -Onix; lane 9 - Zagadca; lane 10 - Peto-86;
lane 11 - Laguna; lane 12 - Nadejda; lane 13 - Novicioc; lane 14 - Leana; lane 15 - 346-I-1; lane 16
- 35-IX-1; lane 17 - 347-IX-1; lane 18 - 351-IV-1; lane 19 - 349-I-2; lane 20 - Dacia; lane 21 - 528;
lane 22 - 13; lane 23 - Lira; lane 24 - 8.
S - susceptible (mi/mi) ; R - resistant (Mi/Mi); R/S - heterozigous (Mi/mi).
Comparative analysis of the molecular and bioassay data demonstrates that the
genotypes Mi/Mi and Mi/mi confer a resistant phenotype (the average scoring range is equal
to or above 0, but below 1.0) for nematode resistance. The screening of twenty two
Moldavian tomato cultivars for resistance to root-knot nematode revealed the perspective of
using in breeding programs of three lines: 35-IX-1 (Mi/mi) , 351-IV-1(Mi/mi), 348-III-2
(Mi/Mi) and one variety Djina (Mi/mi).
Basic PCR techniques are sensitive to changes in reaction conditions that sometimes
make difficult to determine if the absence of an amplification product(s) is due to the
genetic nature of the sample or due to unfavorable reaction conditions. Thus, for the
specificity of analysis it is preferable to use more stable and reliable codominant markers as
Rex-1. Obviously, the application of marker assisted selection is more rapid and convenient
than traditional selection for the trait and can greatly improve the deficiency of tomato
breeding for resistance to root-knot nematodes.
Acknowledgements
We thank Dr. Nadejda Mihnea for kindly providing plant material (Institute of
Genetics, Chisinau, Moldova). The described research was made possible by grants MERL1300 (MRDA, Republic of Moldova and CRDF, USA) and the US National Science
Foundation (grant no. IOB-0543937 to IK).
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