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Proc. Natl. Acad. Sci. USA Vol. 92, pp. 622-625, January 1995 Agricultural Sciences

An aphid-resistance locus is tightly linked to the nematode-resistance gene, Mi, in tomato (Macrosiphum euphorbiae/root-knot nematode/Lycopersicon esculentum)

ISGOUHI KALOSHIAN*, W. HARRY LANGEt, AND VALERIE M. WILLIAMSON*t Departments of *Nematology and tEntomology, University of California, Davis, CA 95616

Communicated by Robert J. Shepherd, University of Kentucky, Lexington, KY, October 11, 1994 (received for review August 2, 1994)

have been reported (13). In this paper we present evidence that potato aphid resistance is conferred by a major gene that resides in close proximity to Mi in tomato.

ABSTRACT Tomato lines from diverse breeding programs were evaluated in the field for resistance to a natural infestation of the potato aphid, Macrosiphum euphorbiae, in Davis, CA. It was noted that all lines that carried the nematode-resistance gene, Mi, displayed aphid resistance. A greenhouse assay for aphid resistance was developed to investigate this relationship. Association of nematode and aphid resistances in near-isogenic lines suggested that these traits are tightly linked. Analysis of an F2 population segregating for nematode resistance indicated that aphid resistance segregated as a single major locus genetically linked to Mi. The name Meul is proposed for this locus. It is likely that Meul was introduced into tomato along with Mi from the wild species Lycopersicon peruvianum. The presence of aphid resistance in the line Motelle, which contains a very small region of introgressed DNA, and the lack of recombinants suggest that Meul is tightly linked to Mi or possibly is the same gene. The map-based strategy currently being used to clone Mi should be applicable to cloning Meul.

MATERIALS AND METHODS Tomato Lines. Tomato lines for field tests were obtained from the sources listed in Table 1. Greenhouse tests for aphid resistance were carried out with Sun6082 and CastlerockII, near-isogenic lines that differ in alleles of Mi, and with Mobox, Motelle, and Mobaci, near-isogenic lines in Moneymaker background (Table 2) (12, 14). An F2 population from a cross of Sun6082 and CastlerockIl was also evaluated in the greenhouse. The nematode-susceptible tomato line UC82 was obtained from Sunseeds Genetics (Hollister, CA), and the nematode-resistant line VFNT cherry was from the C. M. Rick Tomato Genetics Resource Center, University of California, Davis. Field Testing. Tomato breeding lines were evaluated under field conditions for resistance to natural populations of the potato aphid. Plants were started from seed on May 27, 1992, in the Vegetable Crops Department field area on the campus of the University of California, Davis. Tomato lines were planted in 10.5-m-long triply replicated plots arranged in randomized complete block design. The beds were 1.5 m apart. The plots were watered by furrow irrigation weekly. Natural infestations of aphids were counted on August 24-26, 1992. In each plot, 10 complete leaves were selected at random to obtain active colonies of aphids. For each leaf, the leaflet with the greatest number of aphids on the upper and lower surface was selected, and all aphids (apterae and alatae) on the upper and lower surface of this leaflet were counted. The mean number of potato aphids per leaflet was recorded. Greenhouse Screening. A colony of M. euphorbiae (pink form) from a Davis, CA, field population was established in the greenhouse on tomato cultivar UC82. Seeds of plants to be tested were germinated in perlite, and 15-day-old seedlings were transplanted into individual 180-ml cups containing sandy soil. Seedlings were maintained 2 more weeks in a pesticide-free greenhouse with a 16-hr light regime. Plants were moved to a large (122 cm x 84 cm x 84 cm) cage, and aphid-infested UC82 plants were distributed within the cage. Plants were watered with Hoagland's nutrient solution (25) every other day. Greenhouse temperature was maintained at 23-25°C. Twelve days after exposure to aphids, the two most infested leaflets were identified on each plant, and the number of aphids was determined. All forms and sizes were included in the count. Aphid counts were reported as the mean of the two leaflets. REX-1 Marker Analysis. Plant DNA was prepared according to Edwards et al. (15). PCR amplification of the REX-1 marker was carried out with primers and conditions described previously (13). Linkage Analysis. To evaluate linkage between the REX-1 genotype and aphid resistance in the F2 population, regres-

The potato aphid, Macrosiphum eluphorbiae (Thomas), is capable of infesting many plant species. This pest can build up to large numbers on tomato and cause severe damage to some varieties (1-3). Damage includes yield losses caused by stunted growth; shoot dieback; malformation of the leaves; and terminal growth, chlorosis, and necrosis of leaves. Aphids are also efficient virus vectors for many plant viruses. M. euphorbiae is becoming a significant problem in the Sacramento valley area of California, and pesticides are currently being used to control the insect. Because of environmental concern over the use of pesticides in agriculture, alternate means of pest control are being sought. Host-plant resistance is a method of choice for plant-pest suppression. Resistance to potato aphid has been reported in Lycopersicon pennellii Corr (D'Arcy), a wild relative of cultivated tomato Lycopersicon escitlentucm Mill (4). Incorporation of the resistance into tomato has not yet been successful because of the complex inheritance of resistance (5). The resistance is attributed to the sugar esters present in the glandular exudates of type IV trichomes (6). Field observation of a diverse group of tomato lines for resistance to natural infestations of insects revealed a correlation between potato aphid resistance and nematode resistance. It was noted that tomato lines carrying the nematoderesistance gene, Mi, had relatively low numbers of aphids. Mi is a single dominant gene conferring resistance to three species of root-knot nematodes (7, 8). The gene was introduced to cultivated tomato from its wild relative Lycopersicon pernvianuim (9). Isozyme and DNA markers tightly linked to Mi have been identified, and a detailed genetic map of the Mi region has been obtained (10-12). A PCR-based marker, REX-1, tightly linked to Mi, has been developed to easily follow segregation of Mi. No recombinants between REX-1 and Mi The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

ITo whom reprint requests should be addressed. 622

Agricultural Sciences: Kaloshian et aL Table 1. Potato aphid counts on tomato lines Aphids per Mi Source* leaflet,* no. Tomato line genotypet 4 79.6 51.8 mi/mi Omnipak 4 85.0 + 13.6 mi/mi Yuba Gold 4 30.6 14.5 mi/mi FMX 785 4 22.0 7.1 mi/mi FMX 789 4 39.1 10.4 mi/mi FMX 814 4 148.6 + 3.5 FMX 6203 mi/mi 4 22.1 5.7 mi/mi Colusa Gold 2 168.6 67.8 mi/mi Diablo 5 128.6 27.6 mi/mi UC82 L 5 24.0 ± 13.1 mi/mi VF 145 2 mi/mi 29.0 3.8 Murietta 4 104.4 21.6 mi/mi E-6203 6 33.3 + 10.2 mi/mi Casadvance 5 120.9 22.3 mi/mi VF-198 mi/mi 6 38.6 16.4 Shady Lady 72.9 19.7 mi/mi 1 Brigade S 158.2 + 21.8 mi/mi UC82 B 37.2 11.0 7 mi/mi N-4771 4 mi/mi 59.3 14.3 Tracy 2 9.2 1.1 mi/mi Vega 2 mi/mi 55.7 19.9 HMX 8846 104.5 21.0 mi/mi PSR 20512 5 39.6 11.7 mi/mi PSR 20212 124.5 47.6 mi/mi PSR 20112 4 35.2 7.7 mi/mi Apex 100 6 46.4 17.3 mi/mi Castlejay 5 5.6 2.9 Mi/mi Spectrum 151 5 5.4 1.4 Mi/mi Spectrum 385 1.6 Mi/mi 4.3 Spectrum 579 5 3.1 1.2 Mi/mi Nema 1400 5 Mi/mi 11.5 9.1 Nema 1401 2 2.6 ± 1.3 Mi/mi HMX 9851 2 Mi/mi 3.4 ± 1.9 HMX 9849 2 4.9 ± 1.0 Mi/mi Tierra 2 Mi/mi 2.3 ± 2.0 Gemini 4 5.6 ± 2.9 Mi/mi Jackpot 4 5.6 ± 2.9 Mi/mi Monaco 4 3.2 1.5 Mi/mi FMX 154 4 3.0 1.1 Mi/mi FMX 1035 4 3.9 + 1.8 Mi/mi FMX 165 5 2.7 0.7 Mi/mi Zenith 6 2.7 1.0 Mi/mi Sun6066 6 10.4 2.8 Mi/mi Mogambo 2 Mi/Mi 3.9 2.5 Orion 6 13.1 9.7 Mi/Mi Castlerock S 14.9 7.6 Mi/Mi PSR 20412 5 4.2 1.1 Mi/Mi PSR 20312 Tomato lines were evaluated under standard field conditions for resistance to natural populations of the potato aphid. *Average number of aphids per leaflet. For each replicate the number of aphids was counted on the most highly infested leaflet on each of 10 leaves. Each value represents the average of three replicates followed by SEM. tGenotypes Mi/Mi and Mi/mi are nematode resistant; genotype mi/mi is susceptible. *Sources were as follows: 1, Asgrow Seed, San Juan Bautista, CA; 2, Harris Moran, Davis, CA; 3, C. M. Rick Tomato Genetics Resource Center, University of California, Davis; 4, Ferry-Morse Seed, Modesto, CA; 5, PetoSeed, Woodland, CA; 6, Sunseeds Genetics, Hollister, CA; and 7, Rogers N. K., Gilroy, CA. ±

sion analysis was conducted using the general linear model (GLM) procedure in ref. 16.

RESULTS Field Test. Forty-seven tomato lines were evaluated for resistance to natural field infestations of potato aphid (Table

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Table 2. Potato aphid counts on near-isogenic tomato lines for the nematode-resistance gene Mi Mi Aphids,t Nearavg no. genotype: Source§ Tomato line isogenic* A Mi/Mi 1 9.0 ± 2.8 Sun6082 A 1 92.5 ± 6.0 mi/mi CastlerockII B Mi/Mi 2 9.8 ± 5.9 Motelle 2 >100.0 ± 3.3 mi/mi B Mobox B 2 92.5 ± 14.3 mi/mi Mobaci Near-isogenic tomato lines were evaluated in a cage in the greenhouse for resistance to the potato aphid. *Tomato lines with the same letter are near-isogenic or sib lines. tAverage number of aphids per leaflet (two leaflets counted on each of four plants) followed by the SEM. Aphid counts were discontinued once 100 was reached. tGenotype Mi/Mi is nematode resistant; genotype mi/mi is susceptible. §Sources were as follows: 1, Sunseeds Genetics, Hollister, CA; and 2, PetoSeed, Woodland, CA.

1). M euphorbiae occurs in green and pink forms. The nattural infestation in 1992 was primarily the green form. The number of aphids per leaflet ranged from 2.3 to 168.6. In cultivars with more than 100 individuals per leaflet, the aphids caused a stunting of growth, a covering of the plants with white cast skins from molting, and almost complete covering of the fruit with sticky honeydew and secondary sooty black mold. Using the criterion that plants with few aphids (less than 15 per leaflet) are resistant, all lines that contained the Mi gene were resistant to aphids. On all tomato lines lacking Mi except for Vega, the number of aphids per leaflet was greater than 22. Vega was scored as susceptible the following year, suggesting that the low number may have been a sampling error (W.H.L., unpublished data). Greenhouse Screens. To investigate the relationship of the presence of Mi and aphid resistance under controlled conditions, a greenhouse screen was developed. Preliminary experiments with the lines UC82 and VFNT cherry, having susceptible and resistant genotypes, respectively, to root-knot nematodes, revealed that these plants differed significantly in aphid resistance. It was observed that resistance of VFNT cherry increased with age. Five-week old VFNT cherry plants were susceptible, but by 8 weeks, VFNT cherry and UC82 could be easily differentiated. Comparison of the near-isogenic tomato lines Sun6082 and CastlerockIl that differ in nematode resistance was carried out (Table 2). Another set of nearisogenic tomato lines included the susceptible lines Mobaci and Mobox and the resistant line Motelle. In both sets, dramatic differences in the number of aphids correlated with nematode resistance. To confirm genetic linkage of aphid resistance to Mi, an F2 population segregating for Mi was analyzed. Sixty-seven plants were assayed for aphid resistance and for alleles of marker REX-1, which is tightly linked to Mi (13). Plants homozygqus for each of the REX-1 alleles differed dramatically in number of aphids per leaflet (Fig. 1). Heterozygotes exhibited aphid numbers that were intermediate between the two parents, suggesting additive gene action for this presumed gene of aphid resistance. A regression analysis conducted with the segregation class as the independent variable and aphid number as the dependent variable, showed highly significant differences among segregation classes for aphid number (F = 79.2; P = 0.0001). A r2 value of 71.5% suggests the presence of a major gene.

DISCUSSION Field resistance to M. euphorbiae was found to be present in all tomato lines carrying the nematode-resistance gene, Mi. Greenhouse assays on near-isogenic lines and a segregating F2

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REX-I genotype FIG. 1. Average (Av.) number of potato aphids on tomato F2 segregating population and their REX-1 genotype. A total of 67 F2 plants were screened for aphid resistance and REX-1 alleles (p = L. peruvianum, e = L. esculentum) determined. Thirty-nine were heterozygous (e/p) and 14 were homozygous ele andp/p, respectively, for REX-1. Error bars indiate the SEM.

population strongly support the tight linkage of a major gene for aphid resistance with Mi. We propose to call this gene Meul. As far as we are aware, a major resistance gene to potato aphid in tomato has not been reported before. Complexly inherited resistance to the potato aphid associated with the presence of sugar esters in the glandular exudate of the type IV trichomes has been reported in L. pennellii (4, 5, 17). In contrast, the Mi-linked resistance that we describe is simply inherited, and we do not observe any obvious difference in trichomes between resistant and susceptible near-isogenic pairs (data not shown). Field data (Table 1) indicated that lines that do not carry Mi may differ in aphid resistance. If this is true, there may be additional, perhaps independent, resistance factors in domesticated tomato. The linkage of aphid resistance with Mi should make it straightforward for breeders to incorporate aphid resistance into commercial lines. Of course, studies on the breadth of the Meul resistance need to be carried out to assess the value of incorporating this gene. Field data indicate that Meul is a dominant trait, as there was no clear difference between homozygous and heterozygous lines for Mi. In greenhouse tests, the number of aphids on heterozygotes was variable and the average was higher than on homozygous resistant plants, suggesting incomplete dominance. Differences between assays may be due to the confined cage and lack of choice in the greenhouse assay. We hypothesize that Meul was introduced into tomato along with Mi from the wild species L. peruvianum. This wild species has, in fact, been reported to carry resistance to potato aphid (4, 17). The presence of aphid resistance in the Motelle line is particularly compelling as this line retains only about 650 kb of L. peruvianum DNA in the Mi region (refs. 11 and 12; P. Vos, personal communication). Thus, Meul is likely to be within 650 kb of Mi. Major efforts are underway in several laboratories to identify the Mi gene by a map-based cloning approach. The entire introgressed region spanning Mi and Meul has been cloned in yeast artificial chromosome vectors (P. Vos, personal communication). Materials developed to clone Mi should greatly facilitate the cloning of Meul. At this point we have identified no recombinants between Mi and Meul. Thus, it is formally possible that they are the same gene exhibiting pleiotropic effects. We feel that this is

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unlikely as there are nematode isolates that are able to circumvent Mi. Thus, it is unlikely that the recognition mediated by Mi would recognize such a different organism. A more intriguing possibility is that the postulated Meul is a related gene that differs in pest recognition. One can imagine that common resistance mechanisms or pathways of gene induction may be effective for producing resistance to nematodes and aphids. Although aphids and nematodes are unrelated organisms, they do have some common feeding features. Both aphids and root-knot nematodes penetrate their hosts intercellularly. The aphid stylet penetrates and migrates through the plant tissue intercellularly along the middle lamella until it reaches the phloem. Once near the sieve tube the stylet penetrates the cell and removes intracellular fluids (18). Root-knot nematodes penetrate near the root tip and move intercellularly until reaching the vascular bundle where the stylet injects and extracts fluids from the cell (19). Resistance to nematodes mediated by Mi is associated with localized necrosis of cells near or at the feeding site (20, 21). The mechanism of the aphid resistance has not yet been investigated. Further studies are needed to see whether similarities exist between aphid- and nematode-resistance mechanisms. In addition to Meul, the genes Cf-2 and Cf-5 that confer resistance to isolates of the fungus Cladosporiumfulvum Cooke are very tightly linked to Mi (22). Clustering of resistance genes has been postulated to have arisen from recombination/ duplication and rearrangement events to produce novel specificities (23, 24). Clustering of the above resistance genes to taxonomically unrelated plant pests and pathogens is particularly provocative. We thank Dr. P. Gepts for discussions and helpful suggestions on the manuscript. We also thank Drs. C. Rick, D. St Clair, and members of the Williamson lab for critically reading the manuscript and Dr. M. Bauer for help with the statistical analysis. We are grateful to Mr. T. Burlando and G. Miyao for pointing out the presence of aphid resistance in Mi-carrying lines of tomato. This work was supported by U.S. Department of Agriculture Grant 91-37300-6339 and by funds from the California Tomato Research Institute, Inc. 1. Kok-Yokomi, M. L. (1978) Ph.D. Thesis (Univ. of California,

Davis, CA). 2. Lange, W. H. & Bronson, L. (1981) Annu. Rev. Entomol. 26, 345-371. 3. Walker, G. P., Nault, L. R. & Simonet, D. E. (1984) Environ. Entomol. 13, 724-732. 4. Gentile, A. G. & Stoner, A. K. (1968) J. Econ. Entomol. 61, 1152-1154. 5. Goffreda, J. C. & Mutschler, M. A. (1989) Theor. Appl. Genet. 78, 210-216. 6. Goffreda, J. C., Mutschler, M. A., Ave, D. A., Tingey, W. M. & Steffens, J. C. (1989) J. Chem. Ecol. 15, 2135-2147. 7. Gilbert, J. C. & McGuire, D. C. (1956) Proc. Am. Soc. Hortic. Sci. 63, 437-442. 8. Braham, W. S. & Winstead, N. N. (1957) Proc. Am. Soc. Hortic. Sci. 69, 372-377. 9. Smith, P. G. (1944) Proc. Am. Soc. Hortic. Sci. 44, 413-416. 10. Klein-Lankhorst, R., Rietveld, P., Machiels, B., Verkert, R., Wiede, R., Gebhardt, C., Koornneef, M. & Zabel, P. (1991) Theor. Appi. Genet. 81, 661-667. 11. Messeguer, R., Ganal, M., de Vicente, M. C., Young, N. D., Bolkan, H. & Tanksley, S. D. (1991) Theor. Appl. Genet. 82, 529536. 12. Ho, J. Y., Weide, R., Ma, H., van Wordragen, M. F., Lambert, K. N., Koornneef, M., Zabel, P. & Williamson, V. M. (1992) Plant J. 2, 971-982. 13. Williamson, V. M., Ho, J.-Y., Wu, F. F., Miller, N. & Kaloshian, I. (1994) Theor. Appl. Genet. 87, 757-763. 14. Laterrot, H. (1987) Tomato Genet. Coop. Rep. 37, 91. 15. Edwards, K., Johnstone, C. & Thompson, C. (1991) NucleicAcids Res. 19, 1349. 16. SAS Institute (1985) SAS Users Guide: Statistics (SAS Inst., Cary, NC), p. 956.

Agricultural Sciences: Kaloshian et al. 17. Quiros, C. F., Stevens, M. A., Rick, C. M. & Kok-Yokomi, M. L. (1977) J. Am. Soc. Hortic. Sci. 102, 166-171. 18. Pollard, D. G. (1977) inAphids as Virus Vectors, eds. Harris, K. F. & Maramorosch, K. (Academic, New York), pp. 105-118. 19. Linford, M. B. (1937) Phytopathology 27, 824-835. 20. Riggs, R. D. & Winstead, N. N. (1956) Phytopathology 49, 716724.

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21. Rohde, R. A. (1972) Annu. Rev. Phytopathol. 10, 233-252. 22. Dickinson, M. J., Jones, D. A. & Jones, J. D. G. (1993) Mol. Plant-Microbe Int. 6, 341-347. 23. Pryor, T. (1987) Trends Genet. 3, 157-161. 24. Dangl, J. L. (1992) Plant J. 2, 3-11. 25. Lambert, K. N., Tedford, E. C., Caswell, E. P. & Williamson, V. M. (1992) Phytopathology 82, 512-515.