Copyright 0 1995 by the Genetics Society of America

Genomic Localization of Tomato Genes That Control a Hypersensitive Reaction to Xanthomonas campestris pv. vesicatoria (Doidge) Dye

Z. H. YU,*~’ J. F. Wang,t32 R. E. Stallt and C. E. Vallejos”

*Department of Horticultural Sciences, and Plant Molecular and Cellular Biology Group, and tDepartment of Plant Pathology, University of Florida, Gainesvilk, Florida 3261 1

Manuscript received April 27, 1995 Accepted for publication July 7, 1995

ABSTRACT Xunthomonas campestris pv. vesicatoria causes bacterial spot, one of the most serious diseases of tomatoes.

The Lycopersicon esculentum accession ‘Hawaii 7998’ is the only reliable source of resistance to race 1 strains of the pathogen. This resistance is associated with a hypersensitive reaction controlled by multiple nondominant genes. The inoculated area becomes fully necrotic 24 hr after inoculation in ‘Hawaii 7998,’ whereas full necrosis is observed 5 and 4 days after inoculation in the susceptible species L. pennellii (LA 716) and their F,, respectively. An interspecific backcross population, using ‘Hawaii 7998’ as the recurrent parent, was analyzed to determine the linkage relationships between the resistance genes and 135 molecular marker loci. The range of responses of the BC, population included those of the parents. Linkage to a hypersensitive response factor was assessed by comparing the rates of necrosis development between homozygous and heterozygous plants at 8 hr-intervals. Three Factors that affect the hypersensitive response of ‘Hawaii 7998’ were detected. One factor is on the short arm of chromosome 1, another on the long arm of chromosome 1, and a third on the long arm of chromosome 5. These factors appeared to act independently and to have additive effects.

X ANTHOMONAS campestris pv. uesicatoria (Doidge) Dye is the causative agent of bacterial spot in culti-

vated tomatoes ( Zdycopevsicon esculentum) . This pathogen is responsible for one of the most destructive diseases in tropical and subtropical tomato production regions where both high temperature and high relative humid- ity are prevalent. Genetic resistance is the only effective method of control of this disease because cultural prac- tices in combination with antibacterial agents have proven to be unsuccessful. The tomato accession ‘Hawaii 7998’ is the only reliable source of resistance to race 1 strains of the bacterial spot pathogen (SCOTT and JONES 1986); this resistance is associated with a hypersensitive reaction to the pathogen (JONES and SCOTT 1986).

A hypersensitive reaction results from the incompati- ble interaction between an avirulence gene in the patho- gen and a corresponding resistance gene in the plant (FLOR 1956). However, a preliminary genetic analysis of the F,, reciprocal backcrosses and an F2 population between ‘Hawaii 7998’ and L. pennellzi (LA 716), a sus- ceptible wild species, showed that the hypersensitive re- action of ‘Hawaii 7998’ is controlled by more than one

Comsponding author: C . Eduardo Vallejos, Department of Horticul- tural Sciences, 1143 Fifield Hall, University of Florida, Gainesville, FI. 3261 1.

’ Pre.wnl address: USDA/ARS-Southern Plains Area, Southern Crops Research Laboratory, 2765 F & B Rd., College Station, TX 77845. ’ P m m t address: Asian Vegetable Research and Development Cen-

ter, P.O. Box 42, Shanhua, Tainan, Taiwan 74199, Republic of China.

Cenrtics 141: 675-682 ( O c tobc.1-, 1095)

nondominant genetic factor (WANG et al. 1994). The availability of molecular markers that cover the entire tomato genome (TANKSLEY et al. 1992) makes it feasible to subject this complex resistance to genetic analysis. Molecular markers have been used to tag and map dis- ease resistances in many species including tomato (YOUNG et al. 1988; BOURNIVAL et al. 1989, 1990; MARTIN et al. 1991, 1993a), rice (Yu et al. 1991), and maize (MCMULLEN and LOUIE 1989). Furthermore, quantita- tive traits in general, and oligogenic resistances in partic- ular, have also been genetically characterized with mo- lecular markers (PATERSON et al. 1988; CONCIBIDO et d . 1994; DANESH et al. 1994). We report here on the use of DNA markers to probe the tomato genome for the purpose of identiFylng the genes that control the hyper- sensitive reaction of ‘Hawaii 7998’, their chromosome locations, and their possible relationships. An interspe- cific backcross population between ‘Hawaii 7998’ and L. pennellii (LA 716) was chosen for this project. ‘Hawaii 7998’ was used as the recurrent parent because previous segregation analyses of reciprocal backcrosses and F2 progeny indicated that the major components of the hypersensitive reaction were of a nondominant nature (WANG et al. 1994). The L. pennellii accession was se- lected because of its susceptibility to the pathogen and because extensive polymorphisms between this species and L. esculentum can be detected with molecular mark- ers. Low levels of polymorphisms among L. esculentum accessions makes it difficult to follow the segregation of genes in intraspecific crosses. Hybrids between these species show almost normal levels of meiotic pairing

Chromosome Locus hh

1 TG301 66 1 TG334 66 1 TG273 70 1 TG440 38 1 TG157 36 1 TG53 36 1 TG267 35 1 TG27 30 2 TG31 76 2 TG276 79 2 TG306 78 2 TG492 53 2 TG141 70 3 TG525 64 3 TG246 68 3 TGl34 71 3 TG242 67 3 TG2 14 64 3 TG244 64

hP X L (1:1)

39 6.44* 39 6.44* 35 11.01** 63 5.70* 69 9.75** 69 9.75"" 70 11.01** 60 9.34"" 24 26.01** 26 25.75** 27 23.81"" 32 4.71* 35 11.01** 34 8.58"" 32 12.25** 34 12.34** 38 7.42** 41 4.61* 41 4.61*

676 Z. H. Yu et al.

TABLE 1

Marker loci showing significant deviations from Mendelian ratios (1:l) in the backcross progeny

Genotype Genotype

- Chromosome Locus hh hP X 2 ( W

4 TG464 65 33 9.81** 4 TG22 66 39 6.43** 4 TG208 76 29 20.15** 5 TG318 28 70 17.15** 5 TG503 35 70 11.01** 6 TG153 40 65 5.49* 6 TG240 33 72 13.75** 6 TG365 37 68 8.57** 6 TG253 40 65 5.48* 9 TG18 38 67 7.47"* 9 TG328 54 27 8.35**

10 TG52 35 69 10.47** 10 TG596 34 68 10.47** 11 TG194 26 58 11.44** 11 TG47 40 64 5.09" 11 TG400 37 67 8.09** 11 Sod-1 36 66 8.24** 11 TG36 34 69 11.22**

L. pennel6 allele.

and crossing over (KHUSH and RICK 1963). In fact, inter- specific progenies between these species have been used for mapping disease resistances (BOURNWAL et al. 1989, 1990), and in the generation of the current tomato linkage map (TANKSLEY et al. 1992).

MATERIALS AND METHODS

Plant materials: The L. esculentum accession 'Hawaii 7998' and the wild species L. pennellii (LA716) were used as the progenitors of the interspecific backcross population: [L. escu- lentum 'Hawaii 7998' X (L . esculentum 'Hawaii 7998' X L. pen- nellii (LA 716))l. 'Hawaii 7998' has been characterized for its hypersensitive reaction to race 1 strains of X . c. pv. vesicatnria (JONES and SCOTT 1986; WANG et al. 1994). The L. pennellii accession has also been characterized for its susceptibility to the pathogen (WANG et al. 1994). The backcross population consisted of 105 BCI plants. 'Bonny Best,' an extremely sus- ceptible cultivar, was included in all experiments as an addi- tional control.

Bacterial inoculum and disease evaluation: The abaxial side of two opposing leaflets of a fully developed leaf was inoculated by subepidermal injection with lo8 cfu/ml of the X. c. pv. vesicatoria (Xcv) wild strain 90-14. Hypersensitive reactions to Xcv were evaluated after inoculation at 8-hr inter- vals for 96 hr. The development of necrosis in the inoculated area was recorded at the end of each interval. A rating scale of 0-5 was used for this purpose and was based on the propor- tion of the total infiltrated area that had become necrotic at the time of rating. The inoculation protocol was as described in WANG et al. (1994). Bacterial cell counts and the extent of electrolyte leakage have been used to establish the reliability of the rating scale (WmG et al. 1994).

Molecular marker analysis: Isozyme analysis, extraction of plant DNA from frozen leaf tissues, gel electrophoresis, South- ern blotting, hybridizations, and autoradiography were car-

I

*, ** Significant at the 0.05 and 0.01 levels, remectivelv. hh, homozygous for 'Hawaii 7998' alleles; hp, heterozygous with the

ried out essentially as described earlier (VALLEJOS et al. 1992). Total genomic DNA was digested with one of three restriction enzymes: EcnRI, EcnRV, or HindIII. The tomato molecular marker linkage map consists of >lo00 DNA marker loci with an average spacing of - 1.2 cM (TANKSLEY et al. 1992). A subset of these markers, 140 genomic clones, were selected to survey the genome of 'Hawaii 7998.' These markers are evenly spaced throughout the 12 tomato chromosomes. Clones were provided by Dr. STEVE TANKSLEY, Cornell University.

Linkage analysis: The change in necrosis score or rate of necrosis development during each time interval (RN,) was used as the variable to map the genes involved in the hyper- sensitive reaction of 'Hawaii 7998.' All statistical analyses were conducted with the Statistical Analysis System (SAS Institute, SAS Circle, Box 8000, Cary, NC). The computer program MAPMAKER/Exp 3.0 was used for segregation and linkage analysis of molecular marker loci (LANDER et al. 1987; LIN- CO1.N et al. 1992).

RESULTS

Polymorphism and genome coverage: Ninety per- cent of the probes detected polymorphisms between 'Hawaii 7998' and L. pennellii (LA 716). These polymor- phisms were detected with at least one of the three restriction enzymes (EcoRI, EcoRV, or HindIII). Each enzyme detected polymorphisms with 56-67% of the probes. In contrast, restriction fragment length poly- morphisms (RFLPs) between 'Hawaii 7998' and 'Bonny Best', also included in the survey blots, were detected with only 8% of the probes. On average each enzyme detected intraspecific polymorphisms with 3% of the probes; these polymorphisms were scattered through- out the genome.

Resistance to Xanthomonas in Tomato 677

---TG273

- -- r ~ z a

- - - T G ~

“-TG2#5

-- - ~GZM

- - - r ~ m

- - - r~27

--- ~0259

m m d

- - - - r0479

---TG585

llvII

---PrX-7

- - - rot30

- -- r05zs

- -- T G ~

--- rot94

-- - r ~ z 4 z

--- ~ G M Z

---TG&4 - - - TG244 loIol

- -7G549

- -7G94 -

. . . . . . . . . . . .

. . . .

. .

. . . .

. .

. .

. . . . . . . . . . # rDbs T G Z ~

. . affect the hyperscnsitive response.

, .

The segregation of 135 molecular markers (1 1 iso- zymes and 124 RFLPs) was followed in the backcross progeny. Segregation analysis detected significant devi- ations from Mendelian ratios (1:l) with 37 of these markers (Table 1). The distortions were detected in specific regions of nine of the 12 chromosomes. Half of the deviations were due to an excess of homozygotes and the other half due to an excess of heterozygotes. Eight of the deviations were significant at P = 0.05, and the rest were significant at PI 0.01. Deviations detected at low levels of significance were at the boundaries of regions with more severe deviations.

Mapmaker/Exp 3.0 (LINDER PI d . 1987; LINCOLN d al. 1992) was used to determine chromosomal location, gene order, and map distances between the segregating markers of our mapping population. One marker from each chromosome, w i t h complete information, was se- lected as an anchor for that chromosome. The rest of the markers were assigned to their respective chromo- some with the following linkage criteria: a LOD of 4.0 and a maximum distance of 40 cM (Kosambi function). A framework of markers for which a unique position could be determined with a LOD of 2.0 was constructed for each chromosome (Figure 1). Markers that could not be placed in a unique interval were placed on the

FKX:RI< I.-Tomato linkage map constructed with 131 molecular markers. Three chromosome regions carry genes that control the hypersensitive reaction i n ‘Hawaii 7998.’ These genes have been designated m-1 and m-2 on chromosome I and rx-3 on chromosome 5 (W). In addition. on chromosomes 3. 9. and I 1 mark regions of the genome that carry I.. pPnndiialleles that

map next to the nearest marker; these are indicated on the map with small type. A comparison between the map we generated and that published by TANKSLEY Pt

al. (1992) revealed that the probes used in this work covered -95% of the mapped tomato genome. Weak linkage was detected between two markers on the long arm of chromosome 8 (TG33O and TG434). No linkage was detected between two distal markers (TG63 and TG233) on the long arm of chromosome 10 and a markcr on thc short. arm near t.hc ccnt.romcrc (Pm-4). Chromosome location and gene orders in all linkage groups were essentially the same as those originally de- termined by TANKSLEY et al. (1992). At variance were two pairs of markers on chromosome 3 (TG585-Prx- 7) and on chromosome 4 (TC182-TG28), however the inversion in the order occurred over short distances. Striking differences in the recombination values for some chromosome regions were detected. For instance, while we found 23.5 cM between TG295 and TG289 on chromosome I , TANKSLEY d al. (1992) reported 3.4 cM for the same interval.

Hypersensitive response in the parental genotypes and backcross progeny: The time course of‘ necrosis development of ‘Hawdii 7998’ shows an abrupt change in necrosis score during the first hours after inocula-

L. H 678

5

E 4

c 5 2

a

0 0 3 v)

> 1

H7998 I I

I

f F, i

0 16 32 48 64 80 96 112 128

TIME (h)

FIGURE 2.-Timecourse curves of necrosis development after inoculation of 'Hawaii 7998,' L. pennellii (LA 716), and their F,. The necrosis score scale of 0-5 was used. 0, no symptoms; 1, 20% of inoculated area is necrotic; 2, 40%; 3, 60%; 4, 80%; 5, 100% (confluent necrosis).

tion, and confluent necrosis (score of 5 ) is observed within 24 hr (Figure 2). No change in score is possible beyond this point because necrosis development is con- fined to the inoculated area. In contrast, necrosis devel- opment (score change) was not observed in L. pennellii until 5 days after inoculation. WANC et al. (1994) showed that delayed necrosis development is correlated with increased levels of bacterial populations in the inocu- lated area. Development of necrosis in the interspecific hybrid was intermediate but appeared to be closer to the susceptible parent (Figure 2). Inspection of the time course of necrosis development of the backcross population showed variation that covers the entire range of responses: from a fully resistant segregant simi- lar to 'Hawaii 7998' to a fully susceptible segregant simi- lar to L . pennellii (LA 716). The inoculation response of representative segregants that cover the entire spec- trum is shown in Figure 3.

Detection and genomic localization of genes that con- trol the hypersensitive reaction in 'Hawaii 7998': The difference between the responses of the hypersensitive and the susceptible genotypes is in the timing of necrosis score change. For this reason, the timedependent rate of necrosis development (RN,) was calculated for each backcross plant at each &hr interval. These rates were then used as the variable in the genetic analysis of the hypersensitive reaction of 'Hawaii 7998.' The distribu- tion of the rates was discrete because the necrosis rating scale (0-5) allowed for only six possible rates. The fre- quency distribution of the rates was also highly skewed for the majority of the evaluation intervals. This type of data precluded the use of a statistical comparison of genotypic means via t-tests or the use of Mapmaker/ QTL (LANDER and BOTSTEIN 1989). Instead, z-tests were used to compare the genotypic means of the population at each marker locus and at each evaluation interval.

Y U et aL.

5

4 w

0 r 3

0 a 2

1

&""* "" t""t""t" /

0 16 24 32 40 48 56 64 72 80 88 96

TIME (h)

FIGURE 3.-Time-course curves of necrosis development after inoculation of a few selected backcross plants that dis- played the entire range of responses. The same necrosis score scale of 0-5 was used.

Although the rates were not normally distributed, esti- mates of the genotypic means approximate a normal distribution when the sample size is large ( n > 30). We tested the null hypothesis (Ho) that no significant differences exist between the rates of necrosis develop- ment of the two genotypic classes of a given marker

'Hawaii 7998' alleles; hp = heterozygous carrying a 'Hawaii 7998' and a L. pennellii allele). If the test lead to the rejection of the null hypothesis, the alternative hypothesis Ha was assumed and was interpreted as link- age between the marker locus and a gene affecting the rate of necrosis development (Hz,i: ,um(hI , ) > ,uLKNCl,,,) or Ha2: p R N ( l , l , ) < p R N ( l l p ) ) . Ha, is the alternative hypothesis for cases when the homozygous class (hh) has a mean rate that is greater than that of the heterozygous class (hp), whereas Ha2 is the alternative hypothesis for the opposite situation. Hal is the alternative hypothesis for the early evaluation intervals, and Ha2 is the hypothesis for the late evaluation intervals. The rationale for chang- ing the alternative hypothesis with time is derived from the analysis of the responses of the parental genotypes (Figure 2) and those of the segregants (Figure 3). Rates of necrosis development of the resistant genotype ('Hawaii 7998') are greater than zero during the first 24 hr after inoculation, whereas those of the susceptible genotype are zero because necrosis does not develop in this genotype during this period. In contrast, 'Hawaii 7998' rates are zero 80 hr after inoculation because con- fluent necrosis (100%) was reached earlier; however, this is the time when the susceptible genotype begins to develop necrosis in the inoculated area. Accordingly, if a marker locus is linked to a gene involved in the hypersensitive reaction, subtracting the mean rate ofthe heterozygotes from the homozygotes would give a posi- tive value during at least one interval in the first half of the evaluation period and a negative value during at

lOCUS (Ho: /.LRN(i,t,) = pm(hP); hh = homozygous for

Resistance to Xanthomonas in Tomato 679

TABLE 2

Results of >tests used to compare the mean necrosis scores of the genotypic classes: homozygous (hh) vs. heterozygous (hp)

z-statistic

Locus Chromosome 0-16 h 16-24 h 24-32 h 32-40 h 40-48 h 48-56 h 64-72 h 72-80 h 80-88 h 88-96 h

TG301 TG236 TG184 TG24 TG334 TG273 TG440 TG375 TG157 TG53 TG267 TG60 TG351 TG23 TG244 TG549 TG94 TG223

1 I 1 1.68* I 1.75* I I 2.02* I 1 I I I 5 5 2.03* 5 1.97* 3 3 3 9

3.13*** 3.75*** 3.76*** 3.50*** 3.31*** 2.76**

2.33** 1.96* 2.18* 2.33** 2.38** 2.01* 1.84*

TG384 I1 -2.76**

3.93*** 2.80** 3.94*** 2.16* 2.21*

2.65** 3.03** 2.59** 2.37** 1.90* 3.65*** 4.21*** 4.34***

-1.73* -2.16* -1.69 -2.31

-2.54** -2.63**

-3.34*** -3.01** -1.74* -3.67*** -3.84*** -2.69** -2.86**

2.46** -2.67** 2.64**

1.68* 2.39** 2.29*

- 1.88* -2.65** -2.42**

-2.44**

-1.86* -2.23* -2.31*

2.15* 3.03** 1.92* 2.71 2.10 2.09* 2.54**

3.79***

-3.15*** -3.21*** -4.37*** -2.61** - 1.94* -2.85** -4.17*** -3.89*** -4.13*** -4.79*** -3.98*** -3.30*** -3.32*** -3.34***

2.22*

1.93*

*, **, *** Significant at the 0.05, 0.01, and 0.001 levels, respectively. z = (?&, - E,,,)/J[(&/nh),) + ( s ~ n , , , ) ]

least one interval in the second half. Following this ratio- nale we have detected three regions of the tomato ge- nome that contain genes involved in the hypersensitive response of ‘Hawaii 7998’ (Table 2 and Figure 1). One region on the short arm of chromosome 1 is marked by TG301, TG184, TG236, TG24, TG334, and TG273; a second region on the long arm of chromosome I is marked by TG440, TG375, TG157, TG471, TG53, and TG267; and, the third region on chromosome 5 is marked by TG60, TG351, and TG23. The latter region is near the Pto locus (MARTIN et al. 1993a). The Pto gene controls a hypersensitive response to Pseudomonas syn’n- g m pv. tomato and has been recently cloned (MARTIN et al. 1993b). A minimum of one gene per region has been assumed at this time and these genes have been named rx-I, rx-2, and rx3, respectively. These factors can be distinguished not only by their chromosomal location but by their relative strength and the time at which their activities can be detected. The magnitude of their effect can be assessed by inspecting the relative magnitude of the differences of the genotypic means (z-values). rx-1 shows a maximum effect between 16 and 32 hr after inoculation, whereas rx-2 and rx-3 show an effect that peaks during the 2 4 to 32-hr interval; however, the latter appears to have a much stronger effect. The effect of rx-I can be detected over a span of -40 cM, in contrast to the 20 cM over which rx-2 is detected. Unfortunately, a similar comparison is not possible for m-3 due to the absence of markers in the adjacent regions.

Our linkage analysis also revealed that some L. pennel- lii factors had a significant effect on the hypersensitive reaction (Table 2, bottom). In all cases the heterozy- gotes at the marker loci had a significantly higher rate

of necrosis development than the homozygotes during the first half of the evaluation period, while the opposite was detected in the second half. These factors showed linkage to TG244, TG549, and TG94 on chromosome 3, TG223 on chromosome 9, and TG384 on chromo- some 11. The activities of the TG94, TG223-, and TG384linked factors were detected during the 32- to 40-, 2 4 to 32-, and 16 24hr intervals, respectively.

Relationships among the different resistance genes in ‘Hawaii 7998’: The wide range of inoculation responses observed in the backcross population and the differ- ences in the time after inoculation at which each of the three m genes were detected suggest that they act independently. An ad hoc analysis was conducted to test this possibility. The analysis consisted of comparing the genotypic means of the necrosis development rates at one rx-linked marker locus within each of the genotypic classes of the second rx-linked marker locus. The analy- sis showed that the genes located on chromosome 1 appear to act independently (Table 3). The TG236- linked locus (m-I) can be detected in either the homo- zygous or heterozygous groups of the TG157-linked lo- cus (m-2) and vice versa. Furthermore, the effects of the second locus are detected later than those of the first locus as already shown in Table 2. Similar results were obtained with the TG236-linked (rx-I) and TG351- linked (rx-3) locus pair. In contrast, the analysis of the TG157-linked locus (m-2) and the TG351-linked locus (rx-3) indicated the presence of epistasis. The action of one gene cannot be detected when the other is in the homozygous state.

Effect of L. pennellii alleles on the activity of TX genes: Additional ad hoc tests were conducted to determine

680 Z. H. Yu et al.

TABLE 3

Pair-wise Comparisons of necrosis rates of the genotypic classes [homozygous (hh) us. heterozygous (hp)] of one marker locus within one genotypic class (hh or hp) of the second marker locus

z statistic

rx-l - rx-2 - TG236 TG157 TG351 h h h h h h h h h h

rX-3 - 0-16 16-24 24-32 32-40 40-48 48-56 64-72 72-80 80-88 88-96

hh-hp hh-hp

hh hP

hh-hp hh-hp

hh hP

hh hP

hh-hp hh-hp

hh-hp hh-hp

hh hP

hh hP

hh-hp

hh hh-hp

hP hh-hp hh-hp

1.80* 2.70** 2.46** 2.13*

1.94*

3.52*** 2.04* 1.92*

3.33*** 2.41** 2.81**

3.25*** 2.17*

2.59** 2.33** 5.12***

1.86*

1.72*

1.67*

-2.14* -2.69** - 1 .go* -2.33**

-3.24*** -2.72** -2.96** -2.40**

-1.88* -2.18* -2.50** -2.86** -2.19* - 1.85*

-2.61** -2.78** - 1.86*

-2.38** -3.74***

-1.94* -3.40***

*, **, *** Significant at the 0.05, 0.01, and 0.001 levels, respectively. The z statistic was used in these comparisons where =

( ~ t , l l - %p)/J[cs:h/nt,h) + (s:,p/nt,p)l.

whether the L. pennellii alleles that had displayed a sig- nificant effect on the hypersensitive reaction interacted in any way with the three genes from ‘Hawaii 7998’; the results of these tests are shown in Table 4. Each rx locus had a different profile when it was analyzed in conjunction with the L. pennellii alleles. The TG384 linked L. pennellii allele (chromosome 11) significantly increased the hypersensitive reaction of plants that were homozygous at rx-1, rx2 or rx-3, and this effect was also detected in rx-2 heterozygotes. The TG223-linked L. pennellii allele (chromosome 9) significantly increased the hypersensitive response of plants that were homozy- gous or heterozygous at the rx-1 locus; a significant ef- fect was detected only on rx-2 heterozygotes. Finally, a

significant effect of the TG549-linked L. pennellii allele (chromosome 3) was detected only in plants that were heterozygous at either the rx-2 or rx-3 locus.

DISCUSSION

Many disease resistances have been introgressed from the wild species into the cultivated tomato. Molecular marker polymorphisms between the cultivated tomato and the wild species have been exploited to tag and map several disease resistance loci (TANKSLEY 1994). However, the low levels of polymorphisms within L. esculentum (MILLER and TANKSLEY 1990; WILLIAMS and ST. CLAIR 1993) make it difficult to tag and map qualita-

TABLE 4

Pair-wke comparisons of necrosis rates of the genotypic classes [homozygous (hh) vs. heterozygous (hp)] of L. penmZZik derived factors within one genotypic class (hh or hp) of ‘Hawaii 7998’ mlinked loci

z statistic

0-16 16-24 24-32 32-40 40-48 48-56 64-72 72-80 80-88 88-96 1st locus 2nd locus h h h h h h h h h h

~~ ~~

TG236-(rx-I) hh TG223(9) hP

hP

hP

hP

hP

hP

hr,

hh TG384(11)

TG157-(rx-2) hh TG549(3)

hh TG223(9)

hh TG384( 11)

TG351-(m-3) hh TG549(3)

hh TG384( 11)

- 1.80* - 1.92*

-2.86**

- 1.66*

-3.48*** -2.60** - 1.67*

-2.52** 2.59** -2.04*

2.41**

2.68** 1.78*

2.42** 2.87** 1.83*

2.86** 2.31** 3.09***

1.66* 3.21*** 1.91* 2.06*

*, **, *** Significant at the 0.05, 0.01, and 0.001 levels, respectively. The z statistic was used in these comparisons where z =

(Zhh - % p h p ) / h ( S L h h ) + (S;,/nh,)l.

Resistance to Xanthomonas in Tomato 681

tive, and especially quantitative, resistances in intraspe- cific progenies. For instance, in their effort to map an oligogenically controlled resistance to bacterial wilt, DANESH et al. (1994) identified a limited number of useful markers, but only after surveying for polymor- phisms among L. esculentum lines with 16 restriction enzymes. We opted for generating a segregating prog- eny between the source of oligogenic resistance to bac- terial spot, the L. esculentum accession ‘Hawaii 7998,’ and the susceptible wild species L. pennellii (LA 716). This cross facilitated the efficient detection of polymor- phisms between the selected parents with only three restriction enzymes. This efficiency, in turn, ensured a greater genome coverage.

We have used 135 molecular markers (1 1 isozymes and 124 DNA genomic clones) to probe -95% of the mapped tomato genome. Linkage analysis using the rate of necrosis development during the first and sec- ond halves of the 9Ghr evaluation period after inocula- tion revealed the presence of three chromosome re- gions bearing genes that affect the hypersensitive reaction to race 1 strains of X . c. pv. vesicatok. A mini- mum of one gene per region has been assumed at this time. The results presented here indicated that these factors are nondominant and act independently. Al- though dominance/recessiveness relationships can be controlled by the genetic background of the organism (FISHER 1958), the similarity of the responses of inter- and intraspecific (SCOTT and JONES 1989) hybrids of ‘Hawaii 7998’ point to the uniformity of the mode of gene action of the resistance genes in a wide range of genetic backgrounds.

Several lines of evidence strongly suggest that the three genes from ‘Hawaii 7998’ act independently. Among these is the apparent continuous variation of the hypersensitive reaction in the backcross population, and therefore, the lack of two distinct phenotypic classes (one resistant, seven susceptible). The r-tests re- vealed that one locus can be detected regardless of the genotype at a second locus (Table 3), these results in turn revealed that each gene has the following: a unique temporal profile, a different effect on the rate of necro- sis development, and a different interaction profile with the L. pennellikderived genes detected on chromosomes 3, 9, and 11. An exception was the epistatic interactions detected between m-2 and m-3. These results can be explained as follows: the activity of the m-3 gene can be detected earlier than that of the rx-2 gene (Table 2). Development of a high necrosis score in the early hours after inoculation in plants homozygous at the m- 3 can preempt the effect of the m-2 gene. This outcome may be accentuated by the fact that the effect of m-3 appears to be stronger than that of m-2. In addition, the deficiency in the frequency of homozygotes for the chromosome region where m-2 resides (Table 1) has the effect of decreasing the frequency of double homo-

zygotes; this factor, in turn, increases the variance for this class.

Although hypersensitivity-associated resistance is gen- erally controlled by dominant genes, the three genes that control hypersensitivity in ‘Hawaii 7998’ are non- dominant; however, similar cases have been reported for other species ( CRUTE 1985). The presence of three distinct genes that determine an incompatible reaction with a single strain of the pathogen raises the question of whether each gene interacts with a different aviru- lence gene of the pathogen. Only one avirulence gene (avrRxv) from race 1 of this pathogen has been charac- terized and cloned (WHALEN et al. 1993). The availabil- ity of linked molecular markers will facilitate the gener- ation of near isogenic lines for each one of the rx genes; these lines in turn will be useful in the genetic charac- terization of the pathogen; this work may lead to the identification of additional avirulence genes. However, we cannot discount the possibility that all three genes interact with the same avirulence gene of the pathogen, each gene affecting different parts of the host response mechanism. This possibility is supported by the finding that different avrgenes from the cotton pathogen X. c. pv. maluacearum (strain XcmH) do not display specific gene-for-gene interactions with distinct resistance genes from the host (DEFKER et al. 1992), and recent mutant analysis in tomato where it was demonstrated that more than one host gene can interact with the same aviru- lence gene from the pathogen (SALMERON et al. 1994).

Two additional observations are worthy of discussion. First, the detection of L. pennellii dominant genes that had a significant effect on the hypersensitive reaction was unexpected, but not greatly surprising. This situa- tion resembles that of the B gene found in the green fruited species of tomato (RICK and BUTLER 1956). The B gene increases the accumulation of carotenoids in the fruit, but only in combination with genes from the red fruited species. Thus, the mode of gene action of this gene, like the L. pennellii genes in chromosomes 3, 8 and 10, can change with the genetic background. These genes could be exploited in a breeding program. Second, higher rates of recombination in certain areas of the genome were observed for this particular combi- nation of genotypes. This phenomenon could be ex- plored further and eventually exploited for the con- struction of high density maps. These maps are essential for successful positional cloning efforts (MARTIN et al. 1993a,b).

This research was supported in part by U.S. Department of Agricul- ture grant 91-34135-6153. We thank Dr. S. D. TANKSLEY from Cor- nell University for providing the tomato genomic clones. We also thank Drs. L. C. HANNAH and G. A. MOORE for their critical review of the manuscript and for their valuable suggestions. Florida Agricul- tural Experiment Extension Journal Series R-04525.

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