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Introgression of virus-resistance genes into traditional Spanish tomato cultivars (Solanum lycopersicum L.): Effects on yield and quality
Scientia Horticulturae 198 (2016) 183–190
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Introgression of virus-resistance genes into traditional Spanish tomato cultivars (Solanum lycopersicum L.): Effects on yield and quality Fernando Rubio, Aranzazu Alonso, Santiago García-Martínez, Juan J. Ruiz ∗ Departamento Biología Aplicada, Universidad Miguel Hernández, Carretera de Beniel, km 3,2, 03312 Orihuela, Alicante, Spain
a r t i c l e
i n f o
Article history: Received 1 September 2015 Received in revised form 8 November 2015 Accepted 15 November 2015 Keywords: ‘De la Pera’ ‘Muchamiel’ Linkage drag
a b s t r a c t The objective of this study was to determine and quantify the effects of ToMV, TSWV and TYLCV resistance introgressions (determined by Tm-2a, Sw-5 and Ty-1 genes, respectively) on the yield and quality of fresh market tomatoes. Two sets of near isogenic lines (NILs) containing the eight homozygous combinations for the three resistance genes were developed in the backgrounds of the traditional tomato cultivars ‘De la Pera’ and ‘Muchamiel’. These NILs were grown and evaluated in replicated trials in five locations in south-eastern Spain. Across all the analyses performed, the chromosome fragment that consistently had a greater effect was that containing the Ty-1 gene, which conferred resistance to TYLCV. This gene introgression negatively affected all the studied parameters, except for soluble solids content, and its effect was particularly significant on total and commercial yields, which decreased by 50%. This reduction in agricultural yield would only be acceptable when cultivating under high levels of virus infection. The effects of the other introduced fragments (containing Tm-2a and Sw-5) were minor and more variable, increasing or decreasing depending on the trial and the studied parameter. Advanced breeding lines carrying ToMV and TSWV resistance genes in homozygous conditions can be developed without important losses in agronomic and quality characteristics. However, the Ty-1 gene used for TYLCV resistance should only be used in developing cultivars for highly virus-infected areas. © 2015 Elsevier B.V. All rights reserved.
1. Introduction ‘Muchamiel’, ‘De la Pera’, ‘Valenciano’ and ‘Flor de Baladre’ are traditional tomato cultivar types that are very popular in southeastern Spain. They are still being cultivated by local farmers. Little known outside their production area, these tomatoes are highly esteemed by local people due to their excellent organoleptic quality. In local markets, traditional cultivars sell for three to six times the price of the hybrid varieties (Ruiz and García-Martínez, 2009). However, these landraces are severely endangered and even face the risk of loss because of their high susceptibility to several viruses such as those caused by ToMV, TSWV and TYLCV (Picó et al., 2002). Although the presence of these viruses in tomato fields fluctuates greatly from one year to the next, their incidence strongly decreases the benefits obtained by farmers, and even makes the cultivation of the affected landraces non-viable in many areas. Nevertheless, the loss of these landraces would lead to an irreversible loss of genetic diversity. Commercial hybrid varieties with genetic resistance to the abovementioned viruses have been developed, but these resis-
∗ Corresponding author. E-mail address: [email protected] (J.J. Ruiz). http://dx.doi.org/10.1016/j.scienta.2015.11.025 0304-4238/© 2015 Elsevier B.V. All rights reserved.
tance genes have not been introgressed into local varieties, since these varieties represent only a small seed-market share. In 1998, we started a breeding program for the simultaneous introduction of three dominant genes (Tm-2a, Sw-5 and Ty-1) that confer resistance to these three most relevant viruses in south-eastern Spain (ToMV, TSWV and TYLCV, respectively) into ‘Muchamiel’ and ‘De la Pera’ traditional cultivars using markerassisted backcrossing. The genes Tm-2a and Sw-5 come from the wild tomato Solanum peruvianum L., and Ty-1 originated in the Solanum chilense (Dunal) Reiche accession LA1969, another wild tomato species. Tm-2a, Sw-5 and Ty-1 genes have been isolated and characterized by Lanfermeijer et al. (2003), Brommonschenkel et al. (2000) and Verlaan et al. (2013), respectively. Tm-2a and was Sw5 genes were mapped on the chromosome 9, and encode polypeptides which belong to the coiled-coil, nucleotide-bindingARC, leucine-rich repeat (CC-NBS-LRR) class of resistance proteins. Ty-1 is in the short arm of chromosome 6, and codes for a RNAdependent RNA polymerase (RDR), being a completely new class of resistance genes. As a preliminary result of the breeding program, we have obtained promising pre-breeding materials, which have to be further adapted to the specific agroclimatic conditions of different localities (Ruiz and García-Martínez, 2009). The breeding line Muchamiel UMH 1200 (García-Martínez et al., 2011), homozygous
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for the three resistance genes, was the first release produced by this breeding program. Compared with the original landrace, this homozygous breeding line suffers from losses in yield, which are variable depending on the growing conditions. There have been published reports indicating the negative effect of resistance gene introductions, due to the introgressed genes themselves and/or to linkage drag. Tanksley et al. (1998) observed slight reductions in yield and fruit quality in processing tomatoes with ToMV resistance. Lewis et al. (2007) reported reduced yield and quality in tobacco plants containing the N gene (coming from the wild Nicotiana glutinosa L.), which confers resistance to TMV, suggesting the role of linkage drag. S. hirsutum segments containing deleterious alleles in intervals adjacent to alleles containing late blight resistance were found at horticulturally important quantitative trait loci by Brouwer and St Clair (2004). Studying two tomato breeding lines segregating for the Ty-1 gene, Alonso et al. (2008) found that the introgression of this gene was unfavorable for several productive and quality characters. In an open field experiment with ‘De la Pera’ near isogenic lines (NILs), homozygous for one, two or the three introgressed genes, Rubio et al. (2012) found that the introgression of Tm-2a, Sw-5 and Ty-1 genes affected agronomic and quality traits, probably due to other genes introduced along with the resistance genes during backcrossing (linkage drag). The introduction of genes into multiple chromosome regions tends to mask and make it difficult to estimate the effect on agronomic and quality traits. NILs are identical for the entire genome except for the single introgressed region, and they could make it possible to more efficiently estimate the effects of introgressed regions, both individually and/or jointly (Eshed and Zamir, 1995). The development of NIL populations is a demanding task that requires significant time and effort, but it nevertheless allows us to more precisely estimate the effects of the introgression, including interactions (Keurentjes et al., 2007). The main objective of this assay was to determine and quantify the possible effects of ToMV, TSWV and TYLCV resistance introgressions (determined by Tm-2a, Sw-5 and Ty-1 genes, respectively) on the yield and quality of fresh market tomatoes. In attempting to address this question, we have developed two sets of near isogenic lines (NILs) containing all the homozygous combinations for the three resistance genes in the backgrounds of the traditional tomato cultivars ‘De la Pera’ and ‘Muchamiel’. These NILs were grown in replicated trials in five locations in south-eastern Spain and were evaluated for yield and certain quality traits.
2. Materials and methods 2.1. Plant materials The NIL sets for both the ‘De la Pera’ and ‘Muchamiel’ cultivars were developed by selfing two triple heterozygous plants from the UMH tomato breeding program (Fig. 1). These plants were selected from populations developed through 8 and 11 backcrosses, respectively. The NIL populations consisted of two sets of lines, one set for each traditional cultivar. Each line corresponded to one of the eight possible homozygous combinations of the three resistance genes (Tm-2a, Sw-5 and Ty-1). The genotypes are listed in Table 1. The genotype of each line was confirmed using three CAPS markers routinely used in the breeding program in course (To-3, Aps-F2 and CT220) that are linked to the resistance genes (Ruiz and GarcíaMartínez, 2009).
Fig. 1. Breeding scheme for NIL development. The different generations are included in boxes, and successive generations are linked with an arrow. Selfing rounds are depicted as “encircled multiple symbols”. The Marker Assisted Selection (MAS) step indicates that screenings were performed using markers to fix the different genotypes. Rs, RR and ss mean resistant heterozygous, resistant homozygous and susceptible homozygous, respectively. Table 1 The set of Near Isogenic Lines (NILs) used in this assay, with the genotype for each virus resistance gene (RR, resistant homozygous and ss, susceptible homozygous). Muchamiel
Genotype
De la pera
Genotype
Lines
Tm-2a
Ty-1
Sw-5
Lines
Tm-2a
Ty-1
Sw-5
939, 1000 776, 887 916, 972 942, 1002 929 800 879, 891 850, 993
RR RR RR RR ss ss ss ss
RR RR ss ss RR RR ss ss
RR ss ss RR ss RR RR ss
1339, 1385 565, 631 1422 1415 1458 1378, 1413 1487 1278, 1449
RR RR RR RR ss ss ss ss
RR RR ss ss RR RR ss ss
RR ss ss RR ss RR RR ss
2.2. Field experiments The ‘De la Pera’ and ‘Muchamiel’ lines were grown in different locations (Alicante, Spain) and crop systems from 2009 to 2012 (Table 2). The abbreviations for each trial are as follows: P1, P2 and P3 for the ‘De la Pera’ experiments, and M1 and M2 for the ‘Muchamiel’ experiments. In P1–P3 trials, 12 ‘De la pera’ NILs were grown, whereas in M1 and M2 trials 14 ‘Muchamiel’ NILs (Table 1) were grown. The seeds were germinated in greenhouses and transplanted to the field at the appropriate time in each location at standard plant densities (Table 2). The field experiments consisted of 2–4 replications per NIL with 6–8 plants each. Ungrafted plants were grown vertically with a single stem except in the M1 trial, in which the plants were grafted on Beaufort rootstock (De Ruiter) and grown with two stems. At the beginning of the growing season, mild ToMV symptoms were detected in some susceptible plants in some crop cycles. However, symptoms decreased over time and were not detected during harvesting. No TSWV or TYLCV-infected plants were found in any cycle. 2.3. Evaluated traits The number of inflorescences per plant was measured as the number of inflorescences with at least one open flower the day
Density (plant/m2 )
2.50 2.50 2.50
1.35 (2.70 stems/m2 ) 2.70
Plants per plot
10–15 10 6-7
8 7
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before harvest. The number of inflorescences with fruits per plant was measured as the number of inflorescences with at least one visible fruit the day before harvest. The number of fruits per plant was measured as the total fruits harvested per plant. Fruit weight, calculated as the average of all the harvested fruits, was expressed in grams. Commercial yield, based on fruits that were individually harvested at the commercial ripening state, weekly, was expressed in g/plant (only those fruits weighting more than 50 g were considered commercial fruits). Total yield, considered as the weight of all the fruits harvested per plant, was also expressed in g/plant. Soluble solids content (SSC) was measured using 8–12 commercial fully ripe fruits per plot and line. After the fruits had been juiced, the SSC was estimated with an Atago PR-100 digital refractometer, per duplicate, and the results were expressed as ◦ Brix. Titratable acidity (TA) was measured in the same samples used for SSC measurements with a CRISON pHmatic 23 with 0.1 mol L−1 NaOH to pH 8.1. Data were expressed as percentage of citric acid.
Repetition
2 3–4 3
2 2
Cycle and year
April–July 2009 April–August 2010 April–July 2012
April–August 2011 April–August 2011
2.4. Statistical analysis In each trial, mean values for all traits were analyzed using multifactor ANOVA, comparing the RR genotype (resistant) with the ss genotype (susceptible) for each of the three introduced resistance genes (Tm-2a, Sw-5 and Ty-1). To perform a joint analysis of the 3 ‘De la Pera’ trials, the 2 ‘Muchamiel’ trials and the 5 trials together, normalization was accomplished by subtracting the trait-location mean from each value and then dividing by the standard deviation (Tanksley et al., 1998). Probability values for contrasts between genotypes were calculated using the Newman–Keuls test. The percentages of change in trait means between the RR and ss genotypes for each trait were calculated by subtracting the ss mean from the RR mean and dividing by the ss mean, and then multiplying by 100 (Tanksley et al., 1998).
3.1. Analysis of individual trials
a
U.E.S.: University Experimental Station, L.F.: Local farm.
U.E.S. U.E.S. Open field Open field M1 M2 ’Muchamiel’
U.E.S. L.F. U.E.S. Open field Plastic greenhouse Net house P1 P2 P3 ’De la Pera’
Crop system
Trial conditions Code Type
Table 2 General information and growing conditions of the trials.
Locationa
3. Results
Fig. 2 shows the percentages of change for the parameters in each trial. For the Tm-2a gene (Fig. 2) significant differences were found in several parameters for at least one assay. ‘De la Pera’ and ‘Muchamiel’ trials showed different significant effects for the number of inflorescences with fruit per plant: RR genotypes showed a higher number of inflorescences with fruit than ss genotypes for the M1 and M2 trials, while no differences were detected in the P1 and P3 trials. The ss genotypes also showed higher fruit weight values than the RR genotypes in the M1 and M2 trials, while in the P1 and P2 trials, the values were higher for the RR genotypes. Finally, the titratable acidity (TA) was higher in RR genotypes than in ss genotypes in the M1 and M2 trials, while in the P2 and P3 trials, this value was not significant. The values of the effect of this gene ranged from 31.4% for total production in the P1 trial to -15.4% for fruit weight in the M2 trial. The SSC parameter showed significant differences and was higher in the susceptible genotypes except in the P3 and M2 trials, in which no significant differences were found. For the Sw-5 gene (Fig. 2) significant differences were also found in at least one of the trials. Again, different significant effects were found between the ‘De la Pera’ and ‘Muchamiel’ trials for the number of fruits, commercial yield and total yield. In these cases the RR genotypes showed higher values than the ss genotypes in the ‘Muchamiel’ trials. The effects in the ‘De la Pera’ trials, on the other hand, were the opposite. The effect of the TSWV resistance gene measured as percentage of change ranged from 22.3% for number of fruits to −14.1% for TA, both occurring in the M2 trial.
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Fig. 2. The effect of introgressed virus resistance genes on the studied traits for each trial. The percentage changes between the RR and ss genotypes are shown near of the bars. These changes were calculated by subtracting the ss homozygous mean from the RR homozygous mean and dividing by the ss homozygous mean and then multiplying by 100. The values are positive when the RR genotype results were higher than the ss genotype results. Significantly different at P < 0.05 (Newman–Keuls test) values are in grey colored boxes.
In the case of the Ty-1 gene (Fig. 2), significant differences were found for all parameters except for SSC in the P1 and M2 trials. Different significant effects were only found between the ‘De la Pera’ and ‘Muchamiel’ trials for SSC. RR genotypes showed higher values than ss genotypes in the M1 and M2 assays, while in the P2 and P3 trials, the ss genotype scored higher values. For the other characters, and for all the individual tests, the ss genotype values were higher than the RR genotype values. The percentage of change ranged from 8.6% to −55.1% for SSC and total production, respectively, both occurring in the M1 trial. For total and commercial yield, an important decrease were obtained, which were around −50% at all levels in both the ‘De la Pera’ and ‘Muchamiel’ trials. These results clearly indicated the significant negative effect produced by the introgression of the fragment containing the Ty-1 resistance gene. 3.2. Analysis of the ‘De la Pera’ trials Table 3 shows the results of the ANOVA in the ‘De la Pera’ trials (P1–P3) for the three introgressed resistance genes in addition to the percentages of change. For the Tm-2a gene, significant differences between RR and ss genotypes were found in 5 out of the 8 studied characters (number of fruits, fruit weight, commercial production, total production and SSC). For the characters with significant differences, the values of the resistant plants were higher than those of the susceptible ones, except in the case of SSC. The most highly affected characters were commercial yield, total yield and fruit weight per plant, showing percentages of change of 18.6%,
17.7% and 11.7%, respectively. These results could be due to the effect of the slight incidence of the ToMV virus on plants at the beginning of the growing cycle, as previously discussed in materials and methods. For the Sw-5 gene, significant differences between RR and ss genotypes were only found in 3 of the 8 parameters studied (total production, SSC and TA). Resistant homozygous genotypes showed higher values than susceptible homozygous genotypes with respect to all parameters except for SSC. The results suggest that the presence of the fragment containing the Sw-5 allele only produced a slight decrease in the studied parameters, since the effect ranged from −2.7% to −4.7%. This is significantly lower than the ranges obtained for the effects of the Tm-2a and Ty-1 genes. Finally, for the Ty-1 gene, significant differences between the RR and ss genotypes were found for all the studied characters except for SSC, which showed no significant differences. For the majority of the characters with significant differences, the percentage of change values were always negative, indicating that the ss genotypes had greater values than the RR genotypes, ranging from −6.27% for the number of inflorescences with fruits per plant to −51.65% for commercial yield. The chromosome region containing the Ty-1 gene thus clearly produced a greater deleterious effect than the regions containing the Tm-2a and Sw-5 genes. 3.3. Analysis of the ‘Muchamiel’ trials Table 4 shows the results of the ANOVA in the ‘Muchamiel’ trials (M1 and M2) for the three introgressed virus resistance genes. With respect to the Tm-2a gene, significant effects were found between
Table 3 Parameter means (a) for ‘De la Pera’ trials (P1, P2 and P3) for RR and ss genotypes and the three introgressed virus resistance genes. The significance level was obtained from a Multifactor ANOVA test. Percentage change values (b, in parenthesis) were calculated by subtracting the ss mean from the RR mean and dividing by the ss mean, and then multiplying by 100. The values are positive when the RR genotype results were higher than the ss genotype results. The joint analysis was performed with the standardized values for each individual cycle. Number of inflorescences per plant
Number of inflorescences with fruits per plant
Number of fruits per plant
Fruit weight (g/plant)
Commercial yield (g/plant)
Yield (g/plant)
Soluble solids content (SSC) (◦ Brix)
Titratable acidity (g citric acid/100 g)
Tm-2a
RR ss
7.99a 7.83
(+2.0%)b
6.49 6.53
(−0.6%)
35.01* 33.20
(+5.5%)
66* 59
(+11.7%)
1936* 1632
(+18.6%)
2308* 1960
(+17.7%)
4.74* 4.83
(−1.9%)
0.35 0.35
(−0.8%)
Sw-5
RR ss
7.92 7.91
(+0.1%)
6.48 6.54
(−0.9%)
33.68 34.53
(−2.5%)
62 63
(−1.9%)
1747 1821
(−4.1%)
2084* 2185
(−4.6%)
4.72* 4.85
(−2.7%)
0.34* 0.36
(−4.7%)
Ty-1
RR ss
7.58* 8.25
(-8.1%)
6.30* 6.72
(−6.3%)
27.38* 40.84
(−32.9%)
54* 71
(-24.4%)
1163* 2405
(−51.6%)
1468* 2801
(−47.6%)
4.76 4.81
(−0.9%)
0.31* 0.39
(−20.4%)
*
Significantly different at P < 0.05 (Newman–Keuls test).
Table 4 Parameter means (a) for ‘Muchamiel’ trials (M1 and M2) for RR and ss genotypes and the three introgressed virus resistance genes. The significance level was obtained from a Multifactor ANOVA test. Percentage change values (b, in parenthesis) were calculated by subtracting the ss mean from the RR mean and dividing by the ss mean, and then multiplying by 100. The values are positive when the RR genotype results were higher than the ss genotype results. The joint analysis was performed with the standardized values for each individual cycle. Cycles M1M2/ resistance genes
Number of inflorescences per plant
Number of inflorescences with fruits per plant
Number of fruits per plant
Fruit weight (g/plant)
Commercial yield (g/plant)
Yield (g/plant)
Soluble solids content (SSC) (◦ Brix)
Titratable acidity (g citric acid/100 g)
Tm-2a
RR ss
6.89* a 6.55
(+5.2%)b
5.19* 4.86
(+6.8%)
18.97* 15.89
(+19.3%)
138* 162
(−14.8%)
2662 2506
(+6.2%)
2746 2570
(+6.8%)
3.59 3.63
(−1.2%)
0.32* 0.29
(+12.4%)
Sw-5
RR ss
6.83* 6.61
(+3.2%)
5.20* 4.85
(+7.1%)
19.09* 15.77
(+21.1%)
145* 156
(−6.8%)
2763* 2406
(+14.8%)
2848* 2468
(+15.4%)
3.56* 3.66
(−2.8%)
0.29* 0.32
(−10.9%)
Ty-1
RR ss
6.55* 6.90
(−5.1%)
4.73* 5.32
(−11.2%)
13.85* 21.01
(−34.1%)
133* 167
(−20.3%)
1772* 3396
(−47.8%)
1823* 3493
(−47.8%)
3.71* 3.50
(5.9%)
0.27* 0.34
(−21.6%)
*
F. Rubio et al. / Scientia Horticulturae 198 (2016) 183–190
Cycles P1P2P3/resistance genes
Significantly different at P < 0.05 (Newman–Keuls test).
187
(−20.1%) 0.30* 0.37 (+0.8%) 4.32 4.29 (−46.1%) 1706* 3168 (−51.2%) 1365* 2795 (−27.2%) 81* 111 (−31.7%) 22.12* 32.36 Significantly different at P < 0.05 (Newman–Keuls test). *
5.53* 6.04 (−6.0)% 7.09* 7.54 RR ss Ty-1
(−8.4%)
(−7.1%) 0.32* 0.35 (−3.2%) 4.24* 4.38 (+2.7%) 2470 2404 (+3.4%) 2115 2045 (−5.5%) 93* 99 (+4.1%) (+3.4%) 5.88* 5.69 (+1.5%) 7.37 7.26 RR ss Sw-5
27.78* 26.70
(+2.7%) 0.34* 0.33 (−1.7%) 4.27* 4.34 (+10.9%) 2562* 2312 (+11.8%) (+2.4%) 5.85* 5.71 (+3.4%)b 7.44* a 7.19
28.30* 26.18
(+8.1%)
97 95
(+2.7%)
2196* 1964
Soluble solids content (SSC) (◦ Brix) Yield (g/plant) Commercial yield (g/plant) Fruit weight (g/plant) Number of fruits per plant
RR ss
The presence of ToMV in some susceptible plants could be responsible for the slight decreases obtained compared with the resistant plants, which were not infected. In the assays reported by Tanksley et al. (1998), which measured the effect of the introduction of the Tm-2a allele in a processed tomato line, the RR genotypes showed lower values for pH than the ss genotypes, while the ss genotypes showed greater values in total production and in SSC in the fruits harvested when fully ripened and uniformly red in color. The decrease in values that occurred in the RR genotypes could have been caused by the chromosome segment harboring the Tm-2a gene, which possibly contains one or more tightly linked deleterious recessive genes whose negative effects would only be revealed
Tm-2a
4. Discussion
Number of inflorescences with fruits per plant
Table 5 shows the results of the ANOVA of the jointly analyzed ‘De la Pera’ and ‘Muchamiel’ trials (P1, P2, P3, M1 and M2). When analyzing the effects of the Tm-2a gene, significant differences were found between the RR and ss genotypes for all the studied characters except for fruit weight. The most significantly affected characters in the resistant genotypes were commercial yield, total yield and number of fruits per plant, with percentages of change of 11.7%, 10.9% and 8.1%, respectively. For Sw-5 gene, significant differences between RR and ss genotypes were found in 5 out of the 8 studied parameters (number of inflorescences with fruits, number of fruits, fruit weight, SSC and TA). The change percentages were always positive, indicating that the RR genotypes showed greater values than the ss genotypes, except in the case of TA, for which the ss genotype values exceeded the RR genotype values. The change percentages ranged from 3.2% to −7.1%. These values are significantly lower than those obtained for the Tm-2a and Ty-1 genes, suggesting a slight effect of the fragment containing the Sw-5 allele. No significant differences were found in total and commercial yield. Finally, for the Ty-1 gene, significant differences between the RR and ss genotypes were found for all studied characters except for SSC, which showed no significant differences. In the case of the characters with significant differences, the effects of the gene introgression were always negative, indicating that the ss genotypes showed greater values than the RR genotypes.
Number of inflorescences per plant
3.4. Joint analysis of the ‘Muchamiel’ and the ‘De la Pera’ trials
Cycles P1P2P3M1M2/ resistance genes
the RR and ss genotypes in 5 of the 8 studied characters: number of inflorescences, number of inflorescences with fruit per plant, number of fruits, fruit weight, and TA. Except for the fruit weight parameter, RR genotypes showed higher values than ss genotypes. The gene effect ranged from 5.2% for the number of inflorescences to 19.3% for the number of fruits. The most highly affected characters were the number of fruits, TA and fruit weight, with percentages of change of 19.3%, 12.4% and 14.8%, respectively. As previously indicated, the presence of ToMV in some susceptible plants could be responsible for the decreases obtained in the susceptible genotypes. For the Sw-5 gene, different significant effects were found between RR and ss genotypes in all the parameters studied. The RR genotypes always showed greater values than the ss genotypes except in terms of the number of fruits, SSC and TA. The range of variation for the percentages of change in this case ranged from −2.8% for SSC to 21.1% for the number of fruits. Finally, for the Ty1 gene, significant differences were found between the RR and ss genotypes for all the studied characters. The change percentages were always negative, indicating that the ss genotypes showed greater values than the RR genotypes, ranging from −6.27% for the number of inflorescences with fruits per plant to −51.65 % for commercial production. Similar results were observed in the ‘De la Pera’ trials (Table 3).
Titratable acidity (g citric acid/100 g)
F. Rubio et al. / Scientia Horticulturae 198 (2016) 183–190 Table 5 Parameter means (a) for all trials for RR and ss genotypes and the three introgressed virus resistance genes. The significance level was obtained from a Multifactor ANOVA test. Percentage change values (b, in parenthesis) were calculated by subtracting the ss mean from the RR mean and dividing by the ss mean, and then multiplying by 100. The values are positive when the RR genotype results were higher than ss genotype results. The joint analysis was performed with the standardized values for each individual cycle.
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in the homozygous state, according to Tanksley et al. (1998). In a previous field experiment conducted under open air conditions, with a ‘De la pera’ breeding line segregating for the Ty-1 allele, the RR homozygote for Ty-1 also showed significant decreases for several traits. Alonso et al. (2008) found a 38% reduction in yield and an 18% reduction in fruit weight and TA. Again, no significant effect was found for SSC. These results indicate that the introgression of the Ty-1 gene adversely affects agronomic and quality traits, which is probably also due to other genes introduced along with the resistant gene that are not removed during backcrossing (linkage drag). Verlaan et al. (2011) reported the suppression of recombination in the S. chilense region containing the Ty-1 gene due to the occurrence of two chromosomal inversions between S. chilense LA1969 (the donor species of the Ty-1 gene) and Solanum lycopersicum. In another previous experiment conducted under greenhouse conditions with a ‘Muchamiel’ breeding line segregating for the Ty-1 allele, the RR homozygote for Ty-1 showed reduced percentages for several important traits. García-Martínez et al. (2012a) found a 33% reduction in yield, a 17% reduction in fruit weight and a 10% reduction in TA, comparing RR genotypes with ss genotypes, while no significant effects were found for SSC. Again, these results indicate that the introgression of the Ty-1 gene adversely affects agronomic and quality traits (García-Martínez et al., 2012b, 2014, 2015). While in the ‘Muchamiel’ trials significant differences between RR and ss genotypes were found for all parameters, in the ‘De la Pera’ trials differences were only found for total yield, SSC and TA. For the majority of the characters evaluated in the ‘Muchamiel’ trials, the values of the resistant homozygotes were greater than those of the susceptible homozygotes with respect to all evaluated traits except for fruit weight. The opposite occurred for parameters evaluated in the ‘De la Pera’ trials. Nevertheless, results for ‘Muchamiel’ and ‘De la Pera’ trials coincided regarding quality parameters, since susceptible homozygotes showed greater values than resistant homozygotes. This fact suggests that the presence of the fragment containing the Sw-5 allele slightly decreased the values for these parameters. The presence of the fragment containing the Ty-1 allele clearly had a detrimental effect since the percentage of change for the parameters evaluated ranged from −6.1% for number of inflorescences to −51.2 % for commercial production. These values were significantly greater than those obtained for ToMV and TYLCV resistance genes. Similar results were obtained when each cultivar type was analyzed separately, confirming the significant negative effect produced by introducing this type of resistance. The negative effects in some key yield and quality traits associated with the introgression of virus-resistance genes in homozygous state could be eliminated or reduced by using the genetic resistance in heterozygous state (Tanksley et al., 1998). However, this option, which is valid for the F1 hybrid cultivars production, is not a viable option for tomato landraces, because local farmers are in charge of seed production every year. Another option to reduce or eliminate the negative effect of the Ty-1 gene introgression in homozygous state could be the introduction of others genes, as the Ty-2 gene, derived from Solanum habrochaitesderived line H24 (Hanson et al., 2000), the Ty-3 gene (allelic to Ty-1), derived from S. chilense accessions LA2779 and LA1932 (Verlaan et al., 2013), the Ty-4 gene, derived from S. chilense (Ji et al., 2009) or the Ty-5 gene, derived from the cultivar Tyking (Hutton et al., 2012). Recently, Prasanna et al. (2015) reported that the Ty-2 and Ty-3 gene pyramiding produces tomato lines with a high level of resistance to the begomoviruses tested, achieving broad-spectrum resistance. The data from this study do not clarify whether the effects on yield and quality traits are due to Tm-2a, Sw-5 and Ty-1 genes or to the effect of other genes linked to these genes (linkage drag). In the introgression of the viruses resistance genes from wild relatives, even after several backcrossing generations, large segments of wild
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relatives chromosomes surrounding the target genes remain in the breeding lines or cultivars (Young and Tanksley, 1989). Ganal et al. (1989) reported that the Tm-2a gene is located near the centromere of chromosome 9, a region greatly suppressed in meiotic recombination. Verlaan et al. (2011) reported that the occurrence of two chromosomal inversions between S. chilense LA1969 (the donor species of the Ty-1 gene) and S. lycopersicum cause the suppression of recombination in this region of chromosome 6. Taking into account these difficulties, plant transformation methods could be used to bypass linkage drag effects by facilitating delivery of a desired gene into an elite genetic background without flanking sequences from the donor species (Lewis et al., 2007). 5. Conclusions Across all the analyses performed, the chromosome fragment that consistently had a greater effect was that containing the Ty-1 gene, which conferred resistance to TYLCV. This gene introgression negatively affected all the studied parameters except for SSC, and its effect was particularly significant on total and commercial yields, which decreased by 50%. This reduction in agricultural yield would only be acceptable when cultivating under high levels of virus infection. The effects of the other introduced fragments (containing Tm-2a and Sw-5 genes) were minor and more variable, increasing or decreasing depending on the trial and the studied parameter. To our knowledge, no other study has been reported quantifying the effect of the introgression of genetic resistance to TSWV and TYLCV in tomato cultivars. This information could be very useful for tomato breeders. Advanced breeding lines carrying ToMV and TSWV resistance genes in homozygous conditions can be developed without important losses in agronomic and quality characteristics. However, the Ty-1 gene used for TYLCV resistance should only be used in developing cultivars for highly virus-infected areas. Acknowledgements The authors thank J.M. Sánchez and C. Ballester for assisting in analysis, A. Grau for crop practices, and Fabiola Ruiz, Borja Chacón, Candela Teruel, María Jover and Antonio Rosa for collaborating in these studies. This work was partially supported by the Spanish MICINN through the projects AGL2005-03946, AGL2008-03822 and AGL2011-26957. Thanks to Ansley Evans for the language review. The authors wish to thank the anonymous reviewers for helpful comments on the manuscript. References Alonso, A., García-Martínez, S., Arroyo, A., García-Gusano, M., Grau, A., Giménez-Ros, M., Romano, M.E., Valero, M., Ruiz, J.J., 2008. Efecto de la introducción de resistencia a TYLCV (gen Ty-1) en caracteres productivos y de calidad en tomate. Actas de Hortic. 51, 173–174. Brommonschenkel, S.H., Frary, A., Frary, A., Tanksley, S.D., 2000. The broad-spectrum tospovirus resistance gene Sw-5 of tomato is a homolog of the root-knot nematode resistance gene Mi. Mol. Plant Microbe Interact. 13 (10), 1130–1138. Brouwer, D.J., St Clair, D.A., 2004. Fine mapping of three quantitative trait loci for late blight resistance in tomato using near isogenic lines (NILs) and sub-NILs. Theor. Appl. Genet. 108, 628–638. Eshed, Y., Zamir, D., 1995. An introgression line population of Lycopersicon pennellii in the cultivated tomato enables the identification and fine mapping of yield-associated QTL. Genetics 141, 1147–1162. Ganal, M.W., Young, N.D., Tanksley, S.D., 1989. Pulsed field gel electrophoresis and physical mapping of large DNA fragments in the Tm2a region of chromosome 9 in tomato. Mol. Genet. 215, 395–400. García-Martínez, S., Grau, A., Alonso, A., Rubio, F., Valero, M., Ruiz, J.J., 2011. UMH 1200, a breeding line within the Muchamiel tomato type, resistant to three viruses. HortScience 46 (7), 1054–1055. García-Martínez, S., Giménez, M., Alonso, A., Grau, A., Valero, M., Parra, J., Aguilar, A., Gamayo, J.D.D., Ruiz, J.J., 2012a. The introgression of genetic resistance to
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TYLCV into traditional tomato varieties caused similar yield reductions under different growing conditions. Acta Hortic. 935, 149–152. García-Martínez, S., Grau, A., Alonso, A., Rubio, F., Valero, M., Ruiz, J.J., 2012b. UMH 1203, a multiple virus-resistant fresh-market tomato breeding line for open-field conditions. HortScience 47 (1), 1–2. García-Martínez, S., Grau, A., Alonso, A., Rubio, F., Valero, M., Ruiz, J.J., 2014. UMH 1422 and UMH 1415: two De la pera tomato type breeding lines resistant to viruses with reduced linkage drag. HortScience 49 (11), 1465–1466. García-Martínez, S., Grau, A., Alonso, A., Rubio, F., Carbonell, P., Ruiz, J.J., 2015. UMH 916, UMH 972, UMH 1127 and UMH 1139: four freshmarket breeding lines resistant to viruses within the Muchamiel tomato type. HortScience 50 (6), 927–929. Hanson, P.M., Bernacchi, D., Green, S., Tanksley, S.D., Muniyappa, V., Padmaja, A.S., Chen, H.M., Kuo, G., Fang, D., Chen, J.T., 2000. Mapping a wild tomato introgression associated with tomato yellow leaf curl virus resistance in a cultivated tomato line. J. Am. Soc. Hortic. Sci. 125, 15–20. Hutton, S.F., Scott, J.W., Schuster, D.J., 2012. Recessive resistance to tomato yellow leaf curl virus from the tomato cultivar Tyking is located in same region as Ty-5 on chromosome 4. J. Am. Soc. Hortic. Sci. 47, 324–327. Ji, Y., Scott, J.W., Schuster, D.J., Maxwell, D.P., 2009. Molecular mapping of Ty-4, a tomato yellow leaf curl virus resistance locus on chromosome 3 of tomato. J. Am. Soc. Hortic. Sci. 134, 281–288. Keurentjes, J.J.B., Bentsink, L., Alonso-Blanco, C., Hanhart, C.J., Blankestijn-DeVries, H., Effgen, S., Vreugdenhil, D., Koornneef, M., 2007. Development of a near-Isogenic line population of Arabidopsis thaliana and comparison of mapping power with a recombinant inbred line population. Genetics 175, 891–905. Lanfermeijer, F.C., Dijkhuis, J., Sturre, M.J.G., de Haan, P., Hille, J., 2003. Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-22 from Lycopersicon esculentum. Plant Mol. Biol. 52, 1037–1049. Lewis, R.S., Linger, L.R., Wolff, M.F., Wernsman, E.A., 2007. The negative infuence of N-mediated TMV resistance on yield in tobacco: linkage drag versus pleiotropy. Theor. Appl. Genet. 115, 169–178.
Picó, B., Herraiz, J., Ruiz, J.J., Nuez, F., 2002. Widening the genetic basis of virus resistance in tomato. Sci. Hortic. 94 (1–2), 73–89. Prasanna, H.C., Sinha, D.P., Rai, G.K., Krishna, R., Kashyap, S.P., Singh, N.K., Singh, M., Malathi, V.G., 2015. Pyramiding Ty-2 and Ty-3 genes for resistance to monopartite and bipartite tomato leaf curl viruses of India. Plant Pathol. 64, 256–264. Ruiz, J.J., García-Martínez, S., 2009. Tomato varieties ‘Muchamiel’ and ‘De la Pera’ from the southeast of Spain: genetic improvement to promote on-farm conservation, in European landrace: on-farm conservation, management and use. In: Vetelainen, M., Negri, V., Maxted, N. (Eds.), Biodiversity Technical Bulletin no. 15. Bioversity International, Rome, Italy, pp. 171–176. Rubio, F., García-Martínez, S., Alonso, A., Grau, A., Valero, M., Ruiz, J.J., 2012. Introgressing resistance genes into traditional tomato varieties: effects on yield and quality. Acta Hortic. 935, 29–32. Tanksley, S.D., Bernachi, D., BeckBunn, T., Emmatty, D., Eshed, Y., Inai, S., Lopez, J., Petiard, V., Sayama, H., Uhlig, J., Zamir, D., 1998. Yield and quality evaluations on a pair of processing tomato lines nearly isogenic for the Tm2a gene for resistance to the tobacco mosaic virus. Euphytica 99, 77–83. Verlaan, M.G., Szinay, D., Hutton, S.F., de Jong, H., Kormelink, R., Visser, G.F., Scott, J.W., Bai, Y., 2011. Chromosomal rearrangements between tomato and Solanum chilense hamper mapping and breeding of the TYLCV resistance gene Ty-1. Plant J. 68 (6), 1093–1103. Verlaan, M.G., Hutton, S.F., Ibrahem, R.M., Kormelink, R., Visser, R.G.F., Scott, J.W., Jeremy, D., Edwards, J.D., Bai, Y., 2013. The Tomato yellow leaf curl virus resistance genes Ty-1 and Ty-3 are allelic and code for DFDGD-class RNA–dependent RNA polymerases. PLoS Genet. 9, e1003399. Young, N.D., Tanksley, S.D., 1989. RFLP analysis of the size of chromosomal segments retained around the Tm-2 locus of tomato during backcross breeding. Theor. Appl. Genet. 77, 353–359.
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Introgression of virus-resistance genes into traditional Spanish tomato cultivars (Solanum lycopersicum L.): Effects on yield and quality Fernando Rubio, Aranzazu Alonso, Santiago García-Martínez, Juan J. Ruiz ∗ Departamento Biología Aplicada, Universidad Miguel Hernández, Carretera de Beniel, km 3,2, 03312 Orihuela, Alicante, Spain
a r t i c l e
i n f o
Article history: Received 1 September 2015 Received in revised form 8 November 2015 Accepted 15 November 2015 Keywords: ‘De la Pera’ ‘Muchamiel’ Linkage drag
a b s t r a c t The objective of this study was to determine and quantify the effects of ToMV, TSWV and TYLCV resistance introgressions (determined by Tm-2a, Sw-5 and Ty-1 genes, respectively) on the yield and quality of fresh market tomatoes. Two sets of near isogenic lines (NILs) containing the eight homozygous combinations for the three resistance genes were developed in the backgrounds of the traditional tomato cultivars ‘De la Pera’ and ‘Muchamiel’. These NILs were grown and evaluated in replicated trials in five locations in south-eastern Spain. Across all the analyses performed, the chromosome fragment that consistently had a greater effect was that containing the Ty-1 gene, which conferred resistance to TYLCV. This gene introgression negatively affected all the studied parameters, except for soluble solids content, and its effect was particularly significant on total and commercial yields, which decreased by 50%. This reduction in agricultural yield would only be acceptable when cultivating under high levels of virus infection. The effects of the other introduced fragments (containing Tm-2a and Sw-5) were minor and more variable, increasing or decreasing depending on the trial and the studied parameter. Advanced breeding lines carrying ToMV and TSWV resistance genes in homozygous conditions can be developed without important losses in agronomic and quality characteristics. However, the Ty-1 gene used for TYLCV resistance should only be used in developing cultivars for highly virus-infected areas. © 2015 Elsevier B.V. All rights reserved.
1. Introduction ‘Muchamiel’, ‘De la Pera’, ‘Valenciano’ and ‘Flor de Baladre’ are traditional tomato cultivar types that are very popular in southeastern Spain. They are still being cultivated by local farmers. Little known outside their production area, these tomatoes are highly esteemed by local people due to their excellent organoleptic quality. In local markets, traditional cultivars sell for three to six times the price of the hybrid varieties (Ruiz and García-Martínez, 2009). However, these landraces are severely endangered and even face the risk of loss because of their high susceptibility to several viruses such as those caused by ToMV, TSWV and TYLCV (Picó et al., 2002). Although the presence of these viruses in tomato fields fluctuates greatly from one year to the next, their incidence strongly decreases the benefits obtained by farmers, and even makes the cultivation of the affected landraces non-viable in many areas. Nevertheless, the loss of these landraces would lead to an irreversible loss of genetic diversity. Commercial hybrid varieties with genetic resistance to the abovementioned viruses have been developed, but these resis-
∗ Corresponding author. E-mail address: [email protected] (J.J. Ruiz). http://dx.doi.org/10.1016/j.scienta.2015.11.025 0304-4238/© 2015 Elsevier B.V. All rights reserved.
tance genes have not been introgressed into local varieties, since these varieties represent only a small seed-market share. In 1998, we started a breeding program for the simultaneous introduction of three dominant genes (Tm-2a, Sw-5 and Ty-1) that confer resistance to these three most relevant viruses in south-eastern Spain (ToMV, TSWV and TYLCV, respectively) into ‘Muchamiel’ and ‘De la Pera’ traditional cultivars using markerassisted backcrossing. The genes Tm-2a and Sw-5 come from the wild tomato Solanum peruvianum L., and Ty-1 originated in the Solanum chilense (Dunal) Reiche accession LA1969, another wild tomato species. Tm-2a, Sw-5 and Ty-1 genes have been isolated and characterized by Lanfermeijer et al. (2003), Brommonschenkel et al. (2000) and Verlaan et al. (2013), respectively. Tm-2a and was Sw5 genes were mapped on the chromosome 9, and encode polypeptides which belong to the coiled-coil, nucleotide-bindingARC, leucine-rich repeat (CC-NBS-LRR) class of resistance proteins. Ty-1 is in the short arm of chromosome 6, and codes for a RNAdependent RNA polymerase (RDR), being a completely new class of resistance genes. As a preliminary result of the breeding program, we have obtained promising pre-breeding materials, which have to be further adapted to the specific agroclimatic conditions of different localities (Ruiz and García-Martínez, 2009). The breeding line Muchamiel UMH 1200 (García-Martínez et al., 2011), homozygous
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for the three resistance genes, was the first release produced by this breeding program. Compared with the original landrace, this homozygous breeding line suffers from losses in yield, which are variable depending on the growing conditions. There have been published reports indicating the negative effect of resistance gene introductions, due to the introgressed genes themselves and/or to linkage drag. Tanksley et al. (1998) observed slight reductions in yield and fruit quality in processing tomatoes with ToMV resistance. Lewis et al. (2007) reported reduced yield and quality in tobacco plants containing the N gene (coming from the wild Nicotiana glutinosa L.), which confers resistance to TMV, suggesting the role of linkage drag. S. hirsutum segments containing deleterious alleles in intervals adjacent to alleles containing late blight resistance were found at horticulturally important quantitative trait loci by Brouwer and St Clair (2004). Studying two tomato breeding lines segregating for the Ty-1 gene, Alonso et al. (2008) found that the introgression of this gene was unfavorable for several productive and quality characters. In an open field experiment with ‘De la Pera’ near isogenic lines (NILs), homozygous for one, two or the three introgressed genes, Rubio et al. (2012) found that the introgression of Tm-2a, Sw-5 and Ty-1 genes affected agronomic and quality traits, probably due to other genes introduced along with the resistance genes during backcrossing (linkage drag). The introduction of genes into multiple chromosome regions tends to mask and make it difficult to estimate the effect on agronomic and quality traits. NILs are identical for the entire genome except for the single introgressed region, and they could make it possible to more efficiently estimate the effects of introgressed regions, both individually and/or jointly (Eshed and Zamir, 1995). The development of NIL populations is a demanding task that requires significant time and effort, but it nevertheless allows us to more precisely estimate the effects of the introgression, including interactions (Keurentjes et al., 2007). The main objective of this assay was to determine and quantify the possible effects of ToMV, TSWV and TYLCV resistance introgressions (determined by Tm-2a, Sw-5 and Ty-1 genes, respectively) on the yield and quality of fresh market tomatoes. In attempting to address this question, we have developed two sets of near isogenic lines (NILs) containing all the homozygous combinations for the three resistance genes in the backgrounds of the traditional tomato cultivars ‘De la Pera’ and ‘Muchamiel’. These NILs were grown in replicated trials in five locations in south-eastern Spain and were evaluated for yield and certain quality traits.
2. Materials and methods 2.1. Plant materials The NIL sets for both the ‘De la Pera’ and ‘Muchamiel’ cultivars were developed by selfing two triple heterozygous plants from the UMH tomato breeding program (Fig. 1). These plants were selected from populations developed through 8 and 11 backcrosses, respectively. The NIL populations consisted of two sets of lines, one set for each traditional cultivar. Each line corresponded to one of the eight possible homozygous combinations of the three resistance genes (Tm-2a, Sw-5 and Ty-1). The genotypes are listed in Table 1. The genotype of each line was confirmed using three CAPS markers routinely used in the breeding program in course (To-3, Aps-F2 and CT220) that are linked to the resistance genes (Ruiz and GarcíaMartínez, 2009).
Fig. 1. Breeding scheme for NIL development. The different generations are included in boxes, and successive generations are linked with an arrow. Selfing rounds are depicted as “encircled multiple symbols”. The Marker Assisted Selection (MAS) step indicates that screenings were performed using markers to fix the different genotypes. Rs, RR and ss mean resistant heterozygous, resistant homozygous and susceptible homozygous, respectively. Table 1 The set of Near Isogenic Lines (NILs) used in this assay, with the genotype for each virus resistance gene (RR, resistant homozygous and ss, susceptible homozygous). Muchamiel
Genotype
De la pera
Genotype
Lines
Tm-2a
Ty-1
Sw-5
Lines
Tm-2a
Ty-1
Sw-5
939, 1000 776, 887 916, 972 942, 1002 929 800 879, 891 850, 993
RR RR RR RR ss ss ss ss
RR RR ss ss RR RR ss ss
RR ss ss RR ss RR RR ss
1339, 1385 565, 631 1422 1415 1458 1378, 1413 1487 1278, 1449
RR RR RR RR ss ss ss ss
RR RR ss ss RR RR ss ss
RR ss ss RR ss RR RR ss
2.2. Field experiments The ‘De la Pera’ and ‘Muchamiel’ lines were grown in different locations (Alicante, Spain) and crop systems from 2009 to 2012 (Table 2). The abbreviations for each trial are as follows: P1, P2 and P3 for the ‘De la Pera’ experiments, and M1 and M2 for the ‘Muchamiel’ experiments. In P1–P3 trials, 12 ‘De la pera’ NILs were grown, whereas in M1 and M2 trials 14 ‘Muchamiel’ NILs (Table 1) were grown. The seeds were germinated in greenhouses and transplanted to the field at the appropriate time in each location at standard plant densities (Table 2). The field experiments consisted of 2–4 replications per NIL with 6–8 plants each. Ungrafted plants were grown vertically with a single stem except in the M1 trial, in which the plants were grafted on Beaufort rootstock (De Ruiter) and grown with two stems. At the beginning of the growing season, mild ToMV symptoms were detected in some susceptible plants in some crop cycles. However, symptoms decreased over time and were not detected during harvesting. No TSWV or TYLCV-infected plants were found in any cycle. 2.3. Evaluated traits The number of inflorescences per plant was measured as the number of inflorescences with at least one open flower the day
Density (plant/m2 )
2.50 2.50 2.50
1.35 (2.70 stems/m2 ) 2.70
Plants per plot
10–15 10 6-7
8 7
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before harvest. The number of inflorescences with fruits per plant was measured as the number of inflorescences with at least one visible fruit the day before harvest. The number of fruits per plant was measured as the total fruits harvested per plant. Fruit weight, calculated as the average of all the harvested fruits, was expressed in grams. Commercial yield, based on fruits that were individually harvested at the commercial ripening state, weekly, was expressed in g/plant (only those fruits weighting more than 50 g were considered commercial fruits). Total yield, considered as the weight of all the fruits harvested per plant, was also expressed in g/plant. Soluble solids content (SSC) was measured using 8–12 commercial fully ripe fruits per plot and line. After the fruits had been juiced, the SSC was estimated with an Atago PR-100 digital refractometer, per duplicate, and the results were expressed as ◦ Brix. Titratable acidity (TA) was measured in the same samples used for SSC measurements with a CRISON pHmatic 23 with 0.1 mol L−1 NaOH to pH 8.1. Data were expressed as percentage of citric acid.
Repetition
2 3–4 3
2 2
Cycle and year
April–July 2009 April–August 2010 April–July 2012
April–August 2011 April–August 2011
2.4. Statistical analysis In each trial, mean values for all traits were analyzed using multifactor ANOVA, comparing the RR genotype (resistant) with the ss genotype (susceptible) for each of the three introduced resistance genes (Tm-2a, Sw-5 and Ty-1). To perform a joint analysis of the 3 ‘De la Pera’ trials, the 2 ‘Muchamiel’ trials and the 5 trials together, normalization was accomplished by subtracting the trait-location mean from each value and then dividing by the standard deviation (Tanksley et al., 1998). Probability values for contrasts between genotypes were calculated using the Newman–Keuls test. The percentages of change in trait means between the RR and ss genotypes for each trait were calculated by subtracting the ss mean from the RR mean and dividing by the ss mean, and then multiplying by 100 (Tanksley et al., 1998).
3.1. Analysis of individual trials
a
U.E.S.: University Experimental Station, L.F.: Local farm.
U.E.S. U.E.S. Open field Open field M1 M2 ’Muchamiel’
U.E.S. L.F. U.E.S. Open field Plastic greenhouse Net house P1 P2 P3 ’De la Pera’
Crop system
Trial conditions Code Type
Table 2 General information and growing conditions of the trials.
Locationa
3. Results
Fig. 2 shows the percentages of change for the parameters in each trial. For the Tm-2a gene (Fig. 2) significant differences were found in several parameters for at least one assay. ‘De la Pera’ and ‘Muchamiel’ trials showed different significant effects for the number of inflorescences with fruit per plant: RR genotypes showed a higher number of inflorescences with fruit than ss genotypes for the M1 and M2 trials, while no differences were detected in the P1 and P3 trials. The ss genotypes also showed higher fruit weight values than the RR genotypes in the M1 and M2 trials, while in the P1 and P2 trials, the values were higher for the RR genotypes. Finally, the titratable acidity (TA) was higher in RR genotypes than in ss genotypes in the M1 and M2 trials, while in the P2 and P3 trials, this value was not significant. The values of the effect of this gene ranged from 31.4% for total production in the P1 trial to -15.4% for fruit weight in the M2 trial. The SSC parameter showed significant differences and was higher in the susceptible genotypes except in the P3 and M2 trials, in which no significant differences were found. For the Sw-5 gene (Fig. 2) significant differences were also found in at least one of the trials. Again, different significant effects were found between the ‘De la Pera’ and ‘Muchamiel’ trials for the number of fruits, commercial yield and total yield. In these cases the RR genotypes showed higher values than the ss genotypes in the ‘Muchamiel’ trials. The effects in the ‘De la Pera’ trials, on the other hand, were the opposite. The effect of the TSWV resistance gene measured as percentage of change ranged from 22.3% for number of fruits to −14.1% for TA, both occurring in the M2 trial.
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Fig. 2. The effect of introgressed virus resistance genes on the studied traits for each trial. The percentage changes between the RR and ss genotypes are shown near of the bars. These changes were calculated by subtracting the ss homozygous mean from the RR homozygous mean and dividing by the ss homozygous mean and then multiplying by 100. The values are positive when the RR genotype results were higher than the ss genotype results. Significantly different at P < 0.05 (Newman–Keuls test) values are in grey colored boxes.
In the case of the Ty-1 gene (Fig. 2), significant differences were found for all parameters except for SSC in the P1 and M2 trials. Different significant effects were only found between the ‘De la Pera’ and ‘Muchamiel’ trials for SSC. RR genotypes showed higher values than ss genotypes in the M1 and M2 assays, while in the P2 and P3 trials, the ss genotype scored higher values. For the other characters, and for all the individual tests, the ss genotype values were higher than the RR genotype values. The percentage of change ranged from 8.6% to −55.1% for SSC and total production, respectively, both occurring in the M1 trial. For total and commercial yield, an important decrease were obtained, which were around −50% at all levels in both the ‘De la Pera’ and ‘Muchamiel’ trials. These results clearly indicated the significant negative effect produced by the introgression of the fragment containing the Ty-1 resistance gene. 3.2. Analysis of the ‘De la Pera’ trials Table 3 shows the results of the ANOVA in the ‘De la Pera’ trials (P1–P3) for the three introgressed resistance genes in addition to the percentages of change. For the Tm-2a gene, significant differences between RR and ss genotypes were found in 5 out of the 8 studied characters (number of fruits, fruit weight, commercial production, total production and SSC). For the characters with significant differences, the values of the resistant plants were higher than those of the susceptible ones, except in the case of SSC. The most highly affected characters were commercial yield, total yield and fruit weight per plant, showing percentages of change of 18.6%,
17.7% and 11.7%, respectively. These results could be due to the effect of the slight incidence of the ToMV virus on plants at the beginning of the growing cycle, as previously discussed in materials and methods. For the Sw-5 gene, significant differences between RR and ss genotypes were only found in 3 of the 8 parameters studied (total production, SSC and TA). Resistant homozygous genotypes showed higher values than susceptible homozygous genotypes with respect to all parameters except for SSC. The results suggest that the presence of the fragment containing the Sw-5 allele only produced a slight decrease in the studied parameters, since the effect ranged from −2.7% to −4.7%. This is significantly lower than the ranges obtained for the effects of the Tm-2a and Ty-1 genes. Finally, for the Ty-1 gene, significant differences between the RR and ss genotypes were found for all the studied characters except for SSC, which showed no significant differences. For the majority of the characters with significant differences, the percentage of change values were always negative, indicating that the ss genotypes had greater values than the RR genotypes, ranging from −6.27% for the number of inflorescences with fruits per plant to −51.65% for commercial yield. The chromosome region containing the Ty-1 gene thus clearly produced a greater deleterious effect than the regions containing the Tm-2a and Sw-5 genes. 3.3. Analysis of the ‘Muchamiel’ trials Table 4 shows the results of the ANOVA in the ‘Muchamiel’ trials (M1 and M2) for the three introgressed virus resistance genes. With respect to the Tm-2a gene, significant effects were found between
Table 3 Parameter means (a) for ‘De la Pera’ trials (P1, P2 and P3) for RR and ss genotypes and the three introgressed virus resistance genes. The significance level was obtained from a Multifactor ANOVA test. Percentage change values (b, in parenthesis) were calculated by subtracting the ss mean from the RR mean and dividing by the ss mean, and then multiplying by 100. The values are positive when the RR genotype results were higher than the ss genotype results. The joint analysis was performed with the standardized values for each individual cycle. Number of inflorescences per plant
Number of inflorescences with fruits per plant
Number of fruits per plant
Fruit weight (g/plant)
Commercial yield (g/plant)
Yield (g/plant)
Soluble solids content (SSC) (◦ Brix)
Titratable acidity (g citric acid/100 g)
Tm-2a
RR ss
7.99a 7.83
(+2.0%)b
6.49 6.53
(−0.6%)
35.01* 33.20
(+5.5%)
66* 59
(+11.7%)
1936* 1632
(+18.6%)
2308* 1960
(+17.7%)
4.74* 4.83
(−1.9%)
0.35 0.35
(−0.8%)
Sw-5
RR ss
7.92 7.91
(+0.1%)
6.48 6.54
(−0.9%)
33.68 34.53
(−2.5%)
62 63
(−1.9%)
1747 1821
(−4.1%)
2084* 2185
(−4.6%)
4.72* 4.85
(−2.7%)
0.34* 0.36
(−4.7%)
Ty-1
RR ss
7.58* 8.25
(-8.1%)
6.30* 6.72
(−6.3%)
27.38* 40.84
(−32.9%)
54* 71
(-24.4%)
1163* 2405
(−51.6%)
1468* 2801
(−47.6%)
4.76 4.81
(−0.9%)
0.31* 0.39
(−20.4%)
*
Significantly different at P < 0.05 (Newman–Keuls test).
Table 4 Parameter means (a) for ‘Muchamiel’ trials (M1 and M2) for RR and ss genotypes and the three introgressed virus resistance genes. The significance level was obtained from a Multifactor ANOVA test. Percentage change values (b, in parenthesis) were calculated by subtracting the ss mean from the RR mean and dividing by the ss mean, and then multiplying by 100. The values are positive when the RR genotype results were higher than the ss genotype results. The joint analysis was performed with the standardized values for each individual cycle. Cycles M1M2/ resistance genes
Number of inflorescences per plant
Number of inflorescences with fruits per plant
Number of fruits per plant
Fruit weight (g/plant)
Commercial yield (g/plant)
Yield (g/plant)
Soluble solids content (SSC) (◦ Brix)
Titratable acidity (g citric acid/100 g)
Tm-2a
RR ss
6.89* a 6.55
(+5.2%)b
5.19* 4.86
(+6.8%)
18.97* 15.89
(+19.3%)
138* 162
(−14.8%)
2662 2506
(+6.2%)
2746 2570
(+6.8%)
3.59 3.63
(−1.2%)
0.32* 0.29
(+12.4%)
Sw-5
RR ss
6.83* 6.61
(+3.2%)
5.20* 4.85
(+7.1%)
19.09* 15.77
(+21.1%)
145* 156
(−6.8%)
2763* 2406
(+14.8%)
2848* 2468
(+15.4%)
3.56* 3.66
(−2.8%)
0.29* 0.32
(−10.9%)
Ty-1
RR ss
6.55* 6.90
(−5.1%)
4.73* 5.32
(−11.2%)
13.85* 21.01
(−34.1%)
133* 167
(−20.3%)
1772* 3396
(−47.8%)
1823* 3493
(−47.8%)
3.71* 3.50
(5.9%)
0.27* 0.34
(−21.6%)
*
F. Rubio et al. / Scientia Horticulturae 198 (2016) 183–190
Cycles P1P2P3/resistance genes
Significantly different at P < 0.05 (Newman–Keuls test).
187
(−20.1%) 0.30* 0.37 (+0.8%) 4.32 4.29 (−46.1%) 1706* 3168 (−51.2%) 1365* 2795 (−27.2%) 81* 111 (−31.7%) 22.12* 32.36 Significantly different at P < 0.05 (Newman–Keuls test). *
5.53* 6.04 (−6.0)% 7.09* 7.54 RR ss Ty-1
(−8.4%)
(−7.1%) 0.32* 0.35 (−3.2%) 4.24* 4.38 (+2.7%) 2470 2404 (+3.4%) 2115 2045 (−5.5%) 93* 99 (+4.1%) (+3.4%) 5.88* 5.69 (+1.5%) 7.37 7.26 RR ss Sw-5
27.78* 26.70
(+2.7%) 0.34* 0.33 (−1.7%) 4.27* 4.34 (+10.9%) 2562* 2312 (+11.8%) (+2.4%) 5.85* 5.71 (+3.4%)b 7.44* a 7.19
28.30* 26.18
(+8.1%)
97 95
(+2.7%)
2196* 1964
Soluble solids content (SSC) (◦ Brix) Yield (g/plant) Commercial yield (g/plant) Fruit weight (g/plant) Number of fruits per plant
RR ss
The presence of ToMV in some susceptible plants could be responsible for the slight decreases obtained compared with the resistant plants, which were not infected. In the assays reported by Tanksley et al. (1998), which measured the effect of the introduction of the Tm-2a allele in a processed tomato line, the RR genotypes showed lower values for pH than the ss genotypes, while the ss genotypes showed greater values in total production and in SSC in the fruits harvested when fully ripened and uniformly red in color. The decrease in values that occurred in the RR genotypes could have been caused by the chromosome segment harboring the Tm-2a gene, which possibly contains one or more tightly linked deleterious recessive genes whose negative effects would only be revealed
Tm-2a
4. Discussion
Number of inflorescences with fruits per plant
Table 5 shows the results of the ANOVA of the jointly analyzed ‘De la Pera’ and ‘Muchamiel’ trials (P1, P2, P3, M1 and M2). When analyzing the effects of the Tm-2a gene, significant differences were found between the RR and ss genotypes for all the studied characters except for fruit weight. The most significantly affected characters in the resistant genotypes were commercial yield, total yield and number of fruits per plant, with percentages of change of 11.7%, 10.9% and 8.1%, respectively. For Sw-5 gene, significant differences between RR and ss genotypes were found in 5 out of the 8 studied parameters (number of inflorescences with fruits, number of fruits, fruit weight, SSC and TA). The change percentages were always positive, indicating that the RR genotypes showed greater values than the ss genotypes, except in the case of TA, for which the ss genotype values exceeded the RR genotype values. The change percentages ranged from 3.2% to −7.1%. These values are significantly lower than those obtained for the Tm-2a and Ty-1 genes, suggesting a slight effect of the fragment containing the Sw-5 allele. No significant differences were found in total and commercial yield. Finally, for the Ty-1 gene, significant differences between the RR and ss genotypes were found for all studied characters except for SSC, which showed no significant differences. In the case of the characters with significant differences, the effects of the gene introgression were always negative, indicating that the ss genotypes showed greater values than the RR genotypes.
Number of inflorescences per plant
3.4. Joint analysis of the ‘Muchamiel’ and the ‘De la Pera’ trials
Cycles P1P2P3M1M2/ resistance genes
the RR and ss genotypes in 5 of the 8 studied characters: number of inflorescences, number of inflorescences with fruit per plant, number of fruits, fruit weight, and TA. Except for the fruit weight parameter, RR genotypes showed higher values than ss genotypes. The gene effect ranged from 5.2% for the number of inflorescences to 19.3% for the number of fruits. The most highly affected characters were the number of fruits, TA and fruit weight, with percentages of change of 19.3%, 12.4% and 14.8%, respectively. As previously indicated, the presence of ToMV in some susceptible plants could be responsible for the decreases obtained in the susceptible genotypes. For the Sw-5 gene, different significant effects were found between RR and ss genotypes in all the parameters studied. The RR genotypes always showed greater values than the ss genotypes except in terms of the number of fruits, SSC and TA. The range of variation for the percentages of change in this case ranged from −2.8% for SSC to 21.1% for the number of fruits. Finally, for the Ty1 gene, significant differences were found between the RR and ss genotypes for all the studied characters. The change percentages were always negative, indicating that the ss genotypes showed greater values than the RR genotypes, ranging from −6.27% for the number of inflorescences with fruits per plant to −51.65 % for commercial production. Similar results were observed in the ‘De la Pera’ trials (Table 3).
Titratable acidity (g citric acid/100 g)
F. Rubio et al. / Scientia Horticulturae 198 (2016) 183–190 Table 5 Parameter means (a) for all trials for RR and ss genotypes and the three introgressed virus resistance genes. The significance level was obtained from a Multifactor ANOVA test. Percentage change values (b, in parenthesis) were calculated by subtracting the ss mean from the RR mean and dividing by the ss mean, and then multiplying by 100. The values are positive when the RR genotype results were higher than ss genotype results. The joint analysis was performed with the standardized values for each individual cycle.
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F. Rubio et al. / Scientia Horticulturae 198 (2016) 183–190
in the homozygous state, according to Tanksley et al. (1998). In a previous field experiment conducted under open air conditions, with a ‘De la pera’ breeding line segregating for the Ty-1 allele, the RR homozygote for Ty-1 also showed significant decreases for several traits. Alonso et al. (2008) found a 38% reduction in yield and an 18% reduction in fruit weight and TA. Again, no significant effect was found for SSC. These results indicate that the introgression of the Ty-1 gene adversely affects agronomic and quality traits, which is probably also due to other genes introduced along with the resistant gene that are not removed during backcrossing (linkage drag). Verlaan et al. (2011) reported the suppression of recombination in the S. chilense region containing the Ty-1 gene due to the occurrence of two chromosomal inversions between S. chilense LA1969 (the donor species of the Ty-1 gene) and Solanum lycopersicum. In another previous experiment conducted under greenhouse conditions with a ‘Muchamiel’ breeding line segregating for the Ty-1 allele, the RR homozygote for Ty-1 showed reduced percentages for several important traits. García-Martínez et al. (2012a) found a 33% reduction in yield, a 17% reduction in fruit weight and a 10% reduction in TA, comparing RR genotypes with ss genotypes, while no significant effects were found for SSC. Again, these results indicate that the introgression of the Ty-1 gene adversely affects agronomic and quality traits (García-Martínez et al., 2012b, 2014, 2015). While in the ‘Muchamiel’ trials significant differences between RR and ss genotypes were found for all parameters, in the ‘De la Pera’ trials differences were only found for total yield, SSC and TA. For the majority of the characters evaluated in the ‘Muchamiel’ trials, the values of the resistant homozygotes were greater than those of the susceptible homozygotes with respect to all evaluated traits except for fruit weight. The opposite occurred for parameters evaluated in the ‘De la Pera’ trials. Nevertheless, results for ‘Muchamiel’ and ‘De la Pera’ trials coincided regarding quality parameters, since susceptible homozygotes showed greater values than resistant homozygotes. This fact suggests that the presence of the fragment containing the Sw-5 allele slightly decreased the values for these parameters. The presence of the fragment containing the Ty-1 allele clearly had a detrimental effect since the percentage of change for the parameters evaluated ranged from −6.1% for number of inflorescences to −51.2 % for commercial production. These values were significantly greater than those obtained for ToMV and TYLCV resistance genes. Similar results were obtained when each cultivar type was analyzed separately, confirming the significant negative effect produced by introducing this type of resistance. The negative effects in some key yield and quality traits associated with the introgression of virus-resistance genes in homozygous state could be eliminated or reduced by using the genetic resistance in heterozygous state (Tanksley et al., 1998). However, this option, which is valid for the F1 hybrid cultivars production, is not a viable option for tomato landraces, because local farmers are in charge of seed production every year. Another option to reduce or eliminate the negative effect of the Ty-1 gene introgression in homozygous state could be the introduction of others genes, as the Ty-2 gene, derived from Solanum habrochaitesderived line H24 (Hanson et al., 2000), the Ty-3 gene (allelic to Ty-1), derived from S. chilense accessions LA2779 and LA1932 (Verlaan et al., 2013), the Ty-4 gene, derived from S. chilense (Ji et al., 2009) or the Ty-5 gene, derived from the cultivar Tyking (Hutton et al., 2012). Recently, Prasanna et al. (2015) reported that the Ty-2 and Ty-3 gene pyramiding produces tomato lines with a high level of resistance to the begomoviruses tested, achieving broad-spectrum resistance. The data from this study do not clarify whether the effects on yield and quality traits are due to Tm-2a, Sw-5 and Ty-1 genes or to the effect of other genes linked to these genes (linkage drag). In the introgression of the viruses resistance genes from wild relatives, even after several backcrossing generations, large segments of wild
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relatives chromosomes surrounding the target genes remain in the breeding lines or cultivars (Young and Tanksley, 1989). Ganal et al. (1989) reported that the Tm-2a gene is located near the centromere of chromosome 9, a region greatly suppressed in meiotic recombination. Verlaan et al. (2011) reported that the occurrence of two chromosomal inversions between S. chilense LA1969 (the donor species of the Ty-1 gene) and S. lycopersicum cause the suppression of recombination in this region of chromosome 6. Taking into account these difficulties, plant transformation methods could be used to bypass linkage drag effects by facilitating delivery of a desired gene into an elite genetic background without flanking sequences from the donor species (Lewis et al., 2007). 5. Conclusions Across all the analyses performed, the chromosome fragment that consistently had a greater effect was that containing the Ty-1 gene, which conferred resistance to TYLCV. This gene introgression negatively affected all the studied parameters except for SSC, and its effect was particularly significant on total and commercial yields, which decreased by 50%. This reduction in agricultural yield would only be acceptable when cultivating under high levels of virus infection. The effects of the other introduced fragments (containing Tm-2a and Sw-5 genes) were minor and more variable, increasing or decreasing depending on the trial and the studied parameter. To our knowledge, no other study has been reported quantifying the effect of the introgression of genetic resistance to TSWV and TYLCV in tomato cultivars. This information could be very useful for tomato breeders. Advanced breeding lines carrying ToMV and TSWV resistance genes in homozygous conditions can be developed without important losses in agronomic and quality characteristics. However, the Ty-1 gene used for TYLCV resistance should only be used in developing cultivars for highly virus-infected areas. Acknowledgements The authors thank J.M. Sánchez and C. Ballester for assisting in analysis, A. Grau for crop practices, and Fabiola Ruiz, Borja Chacón, Candela Teruel, María Jover and Antonio Rosa for collaborating in these studies. This work was partially supported by the Spanish MICINN through the projects AGL2005-03946, AGL2008-03822 and AGL2011-26957. Thanks to Ansley Evans for the language review. The authors wish to thank the anonymous reviewers for helpful comments on the manuscript. References Alonso, A., García-Martínez, S., Arroyo, A., García-Gusano, M., Grau, A., Giménez-Ros, M., Romano, M.E., Valero, M., Ruiz, J.J., 2008. Efecto de la introducción de resistencia a TYLCV (gen Ty-1) en caracteres productivos y de calidad en tomate. Actas de Hortic. 51, 173–174. Brommonschenkel, S.H., Frary, A., Frary, A., Tanksley, S.D., 2000. The broad-spectrum tospovirus resistance gene Sw-5 of tomato is a homolog of the root-knot nematode resistance gene Mi. Mol. Plant Microbe Interact. 13 (10), 1130–1138. Brouwer, D.J., St Clair, D.A., 2004. Fine mapping of three quantitative trait loci for late blight resistance in tomato using near isogenic lines (NILs) and sub-NILs. Theor. Appl. Genet. 108, 628–638. Eshed, Y., Zamir, D., 1995. An introgression line population of Lycopersicon pennellii in the cultivated tomato enables the identification and fine mapping of yield-associated QTL. Genetics 141, 1147–1162. Ganal, M.W., Young, N.D., Tanksley, S.D., 1989. Pulsed field gel electrophoresis and physical mapping of large DNA fragments in the Tm2a region of chromosome 9 in tomato. Mol. Genet. 215, 395–400. García-Martínez, S., Grau, A., Alonso, A., Rubio, F., Valero, M., Ruiz, J.J., 2011. UMH 1200, a breeding line within the Muchamiel tomato type, resistant to three viruses. HortScience 46 (7), 1054–1055. García-Martínez, S., Giménez, M., Alonso, A., Grau, A., Valero, M., Parra, J., Aguilar, A., Gamayo, J.D.D., Ruiz, J.J., 2012a. The introgression of genetic resistance to
190
F. Rubio et al. / Scientia Horticulturae 198 (2016) 183–190
TYLCV into traditional tomato varieties caused similar yield reductions under different growing conditions. Acta Hortic. 935, 149–152. García-Martínez, S., Grau, A., Alonso, A., Rubio, F., Valero, M., Ruiz, J.J., 2012b. UMH 1203, a multiple virus-resistant fresh-market tomato breeding line for open-field conditions. HortScience 47 (1), 1–2. García-Martínez, S., Grau, A., Alonso, A., Rubio, F., Valero, M., Ruiz, J.J., 2014. UMH 1422 and UMH 1415: two De la pera tomato type breeding lines resistant to viruses with reduced linkage drag. HortScience 49 (11), 1465–1466. García-Martínez, S., Grau, A., Alonso, A., Rubio, F., Carbonell, P., Ruiz, J.J., 2015. UMH 916, UMH 972, UMH 1127 and UMH 1139: four freshmarket breeding lines resistant to viruses within the Muchamiel tomato type. HortScience 50 (6), 927–929. Hanson, P.M., Bernacchi, D., Green, S., Tanksley, S.D., Muniyappa, V., Padmaja, A.S., Chen, H.M., Kuo, G., Fang, D., Chen, J.T., 2000. Mapping a wild tomato introgression associated with tomato yellow leaf curl virus resistance in a cultivated tomato line. J. Am. Soc. Hortic. Sci. 125, 15–20. Hutton, S.F., Scott, J.W., Schuster, D.J., 2012. Recessive resistance to tomato yellow leaf curl virus from the tomato cultivar Tyking is located in same region as Ty-5 on chromosome 4. J. Am. Soc. Hortic. Sci. 47, 324–327. Ji, Y., Scott, J.W., Schuster, D.J., Maxwell, D.P., 2009. Molecular mapping of Ty-4, a tomato yellow leaf curl virus resistance locus on chromosome 3 of tomato. J. Am. Soc. Hortic. Sci. 134, 281–288. Keurentjes, J.J.B., Bentsink, L., Alonso-Blanco, C., Hanhart, C.J., Blankestijn-DeVries, H., Effgen, S., Vreugdenhil, D., Koornneef, M., 2007. Development of a near-Isogenic line population of Arabidopsis thaliana and comparison of mapping power with a recombinant inbred line population. Genetics 175, 891–905. Lanfermeijer, F.C., Dijkhuis, J., Sturre, M.J.G., de Haan, P., Hille, J., 2003. Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-22 from Lycopersicon esculentum. Plant Mol. Biol. 52, 1037–1049. Lewis, R.S., Linger, L.R., Wolff, M.F., Wernsman, E.A., 2007. The negative infuence of N-mediated TMV resistance on yield in tobacco: linkage drag versus pleiotropy. Theor. Appl. Genet. 115, 169–178.
Picó, B., Herraiz, J., Ruiz, J.J., Nuez, F., 2002. Widening the genetic basis of virus resistance in tomato. Sci. Hortic. 94 (1–2), 73–89. Prasanna, H.C., Sinha, D.P., Rai, G.K., Krishna, R., Kashyap, S.P., Singh, N.K., Singh, M., Malathi, V.G., 2015. Pyramiding Ty-2 and Ty-3 genes for resistance to monopartite and bipartite tomato leaf curl viruses of India. Plant Pathol. 64, 256–264. Ruiz, J.J., García-Martínez, S., 2009. Tomato varieties ‘Muchamiel’ and ‘De la Pera’ from the southeast of Spain: genetic improvement to promote on-farm conservation, in European landrace: on-farm conservation, management and use. In: Vetelainen, M., Negri, V., Maxted, N. (Eds.), Biodiversity Technical Bulletin no. 15. Bioversity International, Rome, Italy, pp. 171–176. Rubio, F., García-Martínez, S., Alonso, A., Grau, A., Valero, M., Ruiz, J.J., 2012. Introgressing resistance genes into traditional tomato varieties: effects on yield and quality. Acta Hortic. 935, 29–32. Tanksley, S.D., Bernachi, D., BeckBunn, T., Emmatty, D., Eshed, Y., Inai, S., Lopez, J., Petiard, V., Sayama, H., Uhlig, J., Zamir, D., 1998. Yield and quality evaluations on a pair of processing tomato lines nearly isogenic for the Tm2a gene for resistance to the tobacco mosaic virus. Euphytica 99, 77–83. Verlaan, M.G., Szinay, D., Hutton, S.F., de Jong, H., Kormelink, R., Visser, G.F., Scott, J.W., Bai, Y., 2011. Chromosomal rearrangements between tomato and Solanum chilense hamper mapping and breeding of the TYLCV resistance gene Ty-1. Plant J. 68 (6), 1093–1103. Verlaan, M.G., Hutton, S.F., Ibrahem, R.M., Kormelink, R., Visser, R.G.F., Scott, J.W., Jeremy, D., Edwards, J.D., Bai, Y., 2013. The Tomato yellow leaf curl virus resistance genes Ty-1 and Ty-3 are allelic and code for DFDGD-class RNA–dependent RNA polymerases. PLoS Genet. 9, e1003399. Young, N.D., Tanksley, S.D., 1989. RFLP analysis of the size of chromosomal segments retained around the Tm-2 locus of tomato during backcross breeding. Theor. Appl. Genet. 77, 353–359.