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Crop Science 40:1247-1251 (2000)
© 2000 Crop Science Society of America

CROP BREEDING, GENETICS & CYTOLOGY

Inheritance and Linkage of Two Genes that Confer Resistance to Fusarium Wilt in Chickpea

Mucella Tekeoglua, Abebe Tullua, Walter J. Kaiserb and Fred J. Muehlbauera

a USDA-ARS, 303W Johnson Hall, Washington State University, Pullman, WA 99164-6434 USA
b USDA-ARS, 59 Johnson Hall, Washington State Univ., Pullman, WA 99164-6402 USA

muehlbau{at}wsu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 
Fusarium oxysporum Schlechtend.: Fr. f. sp. ciceris (Padwick) causes a vascular wilt of chickpea (Cicer arietinum L.) and significantly limits production worldwide. The objectives of this study were (i) to determine the inheritance of resistance to races 0 and 5 of fusarium wilt and the genetic map positions of the resistance genes and (ii) to assess the linkage relationships between these two genes and other known wilt resistance genes in chickpea. Seedlings of 131 F6-derived recombinant inbred lines (RILs) were tested for reaction to races 0 and 5. A 1 resistant:1 susceptible segregation ratio was observed for both races, indicating that resistance to each race is controlled by a single gene. Linkage analysis indicated that the genes for resistance to races 4 and 5 were in the same linkage group and were separated by 11.2 centiMorgans (cM). The gene for resistance to race 0 was not linked to the race 4 and 5 resistance genes. In addition, an allele-specific associated primer (ASAP) product (CS-27R/CS-27F), developed from the CS-27 primer, was located between the two resistance genes and was 7.2 and 4 cM from the genes for resistance to races 4 and 5, respectively. Map positions of these two race-specific resistance genes and the marker reported to be linked to the genes for resistance to races 1 and 4 support the hypothesis that wilt resistance genes are clustered on the same chromosome. Since the gene conferring resistance to race 0 is found in a different region of the genome, other genomic regions may be responsible for resistance to wilt pathogens. The gene symbols foc-0, foc-4, and foc-5 are proposed for the genes for resistance to races 0, 4, and 5 of the pathogen, respectively. Identification and further evaluation of disease resistance gene clusters would improve our understanding of wilt resistance in chickpea and facilitate the transfer of resistance genes to new cultivars.

Abbreviations: ASAP, allele-specific associated primer • cM, centiMorgans • ISSR, inter simple sequence repeat • PCR, polymerase chain reaction • RAPD, random amplified polymorphic DNA • RIL, recombinant inbred line


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
Materials and methods
 Results and discussion
 REFERENCES
 
CHICKPEA is the third most important grain legume crop in the world and first in the Mediterranean basin and South Asia (Saxena, 1990; Singh and Ocampo, 1997). Among the biotic stresses that affect chickpea, fusarium wilt caused by F. oxysporum Schlechtend.:Fr. f. sp. ciceris has been reported in many countries as a major yield-limiting factor (Nene and Reddy, 1987; Haware, 1990). The pathogen is either soilborne or seed transmitted (Kraft et al., 1994) and can survive in the soil in the absence of the host for at least 6 yr (Stevenson et al., 1995). The most practical and economical method for controlling the disease is through the use of resistant cultivars.

Seven physiological races (0–6) of the pathogen have been reported (Kraft et al., 1994). The existence of specific races of the pathogen has slowed progress in breeding programs. Haware and Nene (1982) identified races 1 to 4 in India, and later races 0, 5, and 6 were identified in Spain and California (Jimenez-Diaz et al., 1989; Phillips, 1988; Kaiser et al., 1994). Compared with other races of the pathogen, race 0 is the least virulent and causes yellowing symptoms, whereas race 5 is the most virulent and causes severe leaf chlorosis and plant death (Jimenez-Diaz et al., 1991; Kaiser et al., 1994). Although pathogenic races are well established, the genetics of resistance to individual races has not been completely determined.

Upadhyaya et al. (1983a, 1983b) and Singh et al. (1987) reported three independent loci for resistance to race 1 and designated the loci as h1, h2, and h3. A breeding line, WR-315, was defined as resistant, with homozygous recessive genes at all three loci (h1h1h2h2h3h3). A susceptible line, C-104, was characterized as having the genotype H1H1h2h2h3h3 (Singh et al., 1987). Using RILs developed from the cross of these two lines, genes conferring resistance to races 1 and 4 were identified as closely linked to each other and to two random amplified polymorphic DNA (RAPD) markers, CS-27 and UBC-170 (Tullu, 1996). Mayer et al. (1997) developed an ASAP using DNA fragments amplified by the CS-27 RAPD primer to increase the reliability and utility of the marker. Based on these studies, linkage was established between CS-27 and h1, one of the genes for resistance to race 1 (Mayer et al., 1997; Tullu et al., 1998). Also, an inter simple sequence repeat (ISSR) marker linked to the genes for resistance to races 4 and 5 was discovered in the same RIL population used in this study (Ratnaparkhe et al., 1998). The mode of inheritance of other race-specific resistance genes and their locations in the chickpea genome would improve our understanding of the molecular and genetic basis of wilt resistance in chickpea.

In other crops, plant resistance genes appear to be located as a multiple allelic series at a specific locus or as closely linked genes in complex loci. Identification and mapping of these loci on corresponding genomes has been widely demonstrated in a number of crops such as barley (Hordeum vulgare L.) (Giese et al., 1981), maize (Zea mays L.) (Sudupak et al., 1993), flax (Linum usitatissimum L.) (Anderson et al., 1997), and lettuce (Lactuca sativa L.) (Hulbert and Michelmore, 1985).

The objectives of this study were (i) to determine the inheritance of resistance to F. oxysporum f. sp. ciceris races 0 and 5 and the genetic map positions of the resistance genes and (ii) to assess the linkage relationships between these two genes and to other known wilt resistance genes.


   Materials and methods
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
Results and discussion
 REFERENCES
 
One hundred thirty-one RILs, derived from an interspecific cross of C. arietinum (ICC 4958) and C. reticulatum (PI 489777), were advanced by single-seed descent to the F6 in the greenhouse during 1995 to 1996. F8 and F9 seeds of the 131 F6-derived RILs were evaluated for reaction to races 0 and 5 along with the parental lines. ICC-4958 is a wilt-resistant desi type cultivar developed and released by ICRISAT (Saxena et al., 1993), and C. reticulatum (PI 489777) is a susceptible germplasm line from the USDA Regional Plant Introduction Station at Pullman, WA. Cicer reticulatum is considered to be the wild progenitor of C. arietinum and is suitable for use in genetic mapping because of a higher frequency of genetic polymorphism in interspecific crosses when compared with intraspecific crosses within C. arietinum (Gaur and Slinkard, 1990; Simon and Muehlbauer, 1991; Tullu, 1996).

Isolates of Fusarium oxysporum f. sp. ciceris races 0 and 5 used in this study were the same as described by Kaiser et al. (1994). These isolates were used to inoculate the parental lines and a reisolated single spore culture of the pathogen was used to inoculate RILs and parental lines in the experiments.

The pathogen was isolated from fourth-node stem sections taken from infected susceptible parental seedlings according to the procedure described by Tullu et al. (1998). The reisolated pathogen was colonized on filter paper, dried in the transfer hood, and aseptically cut into small pieces. The colonized filter paper pieces were placed in potato-dextrose broth and incubated to produce liquid cultures of the pathogen. The liquid cultures were filtered through four layers of cheesecloth to remove mycelia. The spore suspension was then pelleted by centrifugation at low speed (3000 rpm) for 3 min. After the supernatant was discarded, the conidia were washed with sterile water and the spore suspension was adjusted to 1 x 106 spores mL-1 with a hemacytometer.

Seeds of each RIL and parental line were immersed in metalaxyl [N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester] (Novartis, Greensboro, NC) to control seedborne pathogens. Seeds, 10 to 12 per replication, were then scarified with a lancet and placed in a germination chamber at 22°C and 100% relative humidity for 3 d. Seedlings were then transferred to plastic flats containing sterile coarse perlite in the greenhouse at 21 to 26°C. At the third to fourth nodal stage, seedlings were removed from the perlite and approximately one-third of the root system was pruned as described by Bhatti (1990). The roots of the seedlings were submerged in the inoculum for 5 min, removed from the inoculum, and replanted in the perlite.

The experiments were repeated two or three times for each line and included noninoculated control plants that had been pruned and the roots submerged in distilled water. Disease data were collected at 2-d intervals by counting dead plants during the following 6 to 8 wk for races 5 and 0, respectively. The percentage of surviving plants for each line was used in data analyses. Disease reactions of the RILs were scored as resistant, susceptible, or segregating according to Fisher's least significant difference (P = 0.01). The RILs with surviving plants that exceeded the mean of the susceptible parent plus the LSD value were considered susceptible; RILs with surviving plants that were equal to the resistant parent mean minus the LSD were classified as resistant. Susceptibility of the lines was confirmed by isolating the pathogen from stem sections from the fourth node. For the isolation, 5- to 10-mm-long stem sections were washed in tap water, surface disinfected in 5% bleach for 4 min, and incubated on PDA. Fungi characteristic of F. oxysporum f. sp. ciceris growing from the stem sections was noted and used to verify the scoring.

DNA was isolated from vegetative buds and leaf tissue of the parental lines and RILs using the miniprep technique of Doyle and Doyle (1987). DNA was used for polymerase chain reactions (PCR) following the protocol as described in Mayer et al. (1997). Allele-specific associated primers for CS-27700 and UBC-170550 and an ISSR primer (UBC-855) were used in PCR amplifications. The PCR products were separated on 2% (w/v) agarose gels, stained with ethidium bromide, and scored for presence or absence of bands.

Because of limitations of greenhouse space, screening of the RILs for resistance to race 0 was conducted in seven experiments with 38 lines including parents in each experiment. Scoring for race 5 resistance was carried out with 35 lines in each of eight experiments. Analysis of variance for the experiments was performed using the SAS statistical software (SAS Institute, Cary, NC) Proc Mixed procedure where experiment was considered as a random factor and RIL as a fixed factor. The RILs were evaluated in either two or three experiments and all the data were considered in the analyses. Means were separated by Fisher's least significant difference (LSD) (P = 0.01). Goodness of fit to expected segregation ratios were determined by {chi}2 analysis.

Reaction of the RILs to race 4 and data for UBC-855 were provided by Tullu (1996) and Ratnaparkhe et al. (1998), respectively. The Mapmaker computer program (Lander et al., 1987) was used to determine recombination frequencies and locus order.


   Results and discussion
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
REFERENCES
 
Resistance to Race 0
Although Fusarium oxysporum f. sp. ciceris race 0 is characterized as inducing foliar yellowing syndrome, we observed wilting reactions similar to those induced by race 5 on C. reticulatum (PI 489777) and susceptible RILs. Cicer reticulatum died within 30 d of inoculation with race 0, whereas ICC-4958 was resistant.

Differences among parental lines and resistant and susceptible RILs were highly significant (Table 1) . All ICC-4958 seedlings survived in all replications. Survival of resistant RILs ranged from 76 to 100% and susceptible RILs had 0 to 26% survival, which was similar to the susceptible parent C. reticulatum (11% survival) (Fig. 1) . Because there was no variation for the resistant parent's scores, we used variance of the susceptible parent mean to calculate the LSD value of 15% (P = 0.01).


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  		Table 1 Analysis of variance of chickpea parental lines and recombinant inbred lines (RILs) for reaction to races 0 and 5 of Fusarium oxysporum f. sp. ciceri

 


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  		Fig. 1 Frequency distribution of recombinant inbred lines (RILs) segregating for resistance to Fusarium oxysporum f. sp. ciceris race 0. Resistant (R) and susceptible (S) chickpea parent means are indicated by arrows

 
Because of limited seed, five of the RILs could not be evaluated. Of the remaining 126 RILs, 64 were resistant, 56 were susceptible, and six were segregating. The data closely fit a 1:1 segregation ratio, indicating that resistance is controlled by a single gene (Table 2) . The number of RILs that were still segregating was similar to that expected in the F6. Since the RILs were F6-derived, we assumed that each of these segregating lines came from a plant that was heterozygous at the wilt resistance gene locus in the F6 and segregated in the two proceeding generations. If resistance is controlled by a recessive gene, the expected ratio would approach 10 susceptible:6 resistant in the F8. Segregating lines gave a total of 65 susceptible and 50 resistant plants, which closely fits the expected 10:6 ratio ({chi}2 = 1.757, P = 0.184). We also confirmed the recessive nature of resistance by inoculating F1 seedlings. Since all four F1 plants died after inoculation, a single recessive gene apparently confers resistance.


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  		Table 2 Single locus goodness of fit of the F6 derived chickpea RILs to a 1 susceptible:1 resistant segregation ratio for reaction to two races of the fusarium wilt pathogen and the markers

 
Resistance to Race 5
The ICC-4958 parent was completely resistant (100% survival) to race 5 of the pathogen. Wilt symptoms developed earlier in response to race 5 than race 0 on the susceptible parent (PI 489777) and the RILs. All susceptible plants died within 20 d of inoculation. A highly significant difference was found between susceptible and resistant RILs (Table 1). As with race 0 screening, the resistant parent had 100% survival following inoculation with race 5 and C. reticulatum had from 0 to 25% survival. Reactions of RILs were differentiated using the susceptible parent mean (22% survival) and a LSD value of 21% (P = 0.01) that was calculated based on the variance of the susceptible parent mean. Survival of susceptible and resistant RILs ranged from 0 to 33% and 67 to 100%, respectively (Fig. 2) .



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  		Fig. 2 Frequency distribution of RILs segregating for resistance to Fusarium oxysporumf. sp. ciceris race 5. Resistant (R) and susceptible (S) chickpea parent means are indicated by arrows

 
One hundred twenty-one RILs were evaluated for reaction to race 5. Ten of the RILs could not be included in the analysis because of limited seed. Of the 121 RILs, 53 were resistant, 62 were susceptible, and six segregated. The data closely fit a 1:1 segregation ratio, indicating that resistance to race 5 is controlled by a single gene (Table 2). The six segregating lines had 84 susceptible and 63 resistant plants. As with race 0, the data fit the expected 10 susceptible:6 resistant ratio ({chi}2 = 1.80, P = 0.179) indicating that resistance was conferred by a recessive gene. When F1 plants from this cross were inoculated with race 5 they were completely susceptible, confirming the recessive nature of the resistance gene.

In this study, we were able to identify single recessive genes that confer resistance to races 0 and 5 of fusarium wilt in chickpea. We designated these two genes as foc-0 for resistance to race 0 and foc-5 for resistance to race 5. We also designated the gene identified by Tullu (1996) for resistance to race 4 as foc-4. Recessive race-specific resistance genes have also been reported in tomato (Lycopersicon esculentum Mill.) (Ori et al., 1997) and soybean [Glycine max (L.) Merr.] (Kosslak et al., 1996). However, we do not know which mechanism at the cellular level prevents the pathogen from causing disease on resistant RILs.

During parental and RIL screening, we were able to isolate the pathogen from all susceptible RILs and from C. reticulatum. Fusarium oxysporum f. sp. ciceris was isolated from some resistant lines that showed mild wilt symptoms. To determine the resistance reaction of these lines, we inoculated additional seedlings from each line with the pathogen isolated from that particular line. In most cases, the lines had similar or completely resistant reactions in the second inoculation. Kaiser et al. (1994) reported isolating the wilt pathogen from otherwise resistant plants. This may have been possible due to the high inoculum level used in these studies or due to a hypersensitive reaction of some of the lines. Although race 0 of the pathogen was reported to be the least virulent of the seven races, inducing foliar yellowing symptoms (Kraft et al., 1994), we observed wilt reactions similar to those induced by race 5 on C. reticulatum and susceptible RILs. The same observation was reported in screening of C. reticulatum accessions for reaction to race 0 (Kaiser et al., 1994). Cicer reticulatum was highly susceptible, with symptoms similar to those induced by race 5, the most virulent race.

We were able to identify lines with resistance to one race of fusarium wilt but susceptibility to another race, which suggests different resistance genes confer resistance to different races. These lines could be used as race differentials to facilitate identification of races based on pathogen x host interactions. Identification of variability within the pathogen population could advance our understanding of the resistance mechanism in the host.

Markers Linked to Resistance Genes
One ASAP marker (CS-27) and one RAPD marker, UBC-170, previously shown to be associated with the race 1 and race 4 resistance genes in a different population (Mayer et al., 1997) were evaluated in this population. UBC-170 failed to detect polymorphism between the parents. CS-27 segregated 64:59 (present/absent) ratio, which closely fit a 1:1 segregation ratio (Table 2). Although it was not reported, UBC-855 has been determined to be a marker with distorted segregation (Table 2). Linkage analysis of the markers and the resistance genes showed that foc-4 and foc-5 were separated by 11.2 cM on the same linkage group (Fig. 3) . CS-27 was located between the two resistance genes at 7.2 cM from foc-4 and 4 cM from foc-5. The foc-0 segregated independently of these genes and the CS-27 marker.



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  		Fig. 3 Position of the fusarium wilt resistance genes and the markers on a partial chickpea linkage map. Data in parentheses are from Tullu et al. (1998). Map distances are in centiMorgans

 
According to Tullu et al. (1998), foc-4 and h1 are located 5 cM apart. The gene for resistance to race 4, foc-4, was 9 cM from CS-27 and UBC-170, while the gene for resistance to race 1, h1, was 14 cM from the markers. That CS-27 is linked to foc-4 and foc-5 in this study indicates that h1 could be located in the same region of the genome as foc-4 and foc-5 (Fig. 3). Studies on the genetic fine structure of these linked genes will help enhance our understanding of wilt resistant gene clusters in chickpea. The lack of linkage between foc-0 and other genes conferring wilt resistance indicates that other regions of the genome also are involved in wilt resistance. Clusters of disease resistance genes have been documented in many plant species. These gene clusters have been identified by segregation analysis of resistance genes or molecular markers linked to the genes (Michelmore, 1995). In flax, genes conferring resistance to flax rust occur at five loci as multiple alleles (Flor, 1956). In barley, the Ml-a locus has at least 13 alleles for resistance to powdery mildew [Erysiphe graminis (DC) Merat f.sp. hordei] (Giese, 1981). The Rp1 locus for rust resistance in maize (Sudupak et al., 1993) and the Cf genes in tomato (Parniske et al., 1997) are examples of multiple linked resistance specificities. Also, clusters of genes for resistance to taxonomically diverse pathogens have been identified in lettuce (Kesseli et al., 1993).

Fine structure genetic mapping of the linked genes should be conducted to provide a means for map-based cloning of the wilt resistance genes and studies of their structure and mode of action. Inheritance information for the resistance genes will aid breeding programs aimed at transferring wilt resistance to new cultivars.


   ACKNOWLEDGMENTS
 
We thank Dr. Clarice C. Coyne for her help and support during the course of this study.

Received for publication October 8, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 




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