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The Journal of Heredity 2002:93(4)
© 2002 The American Genetic Association 93:291-293

Brief Communication

Genomic Location of the Fw Gene for Resistance to Fusarium Wilt Race 1 in Peas

M. J. Grajal-Martìn, and F. J. Muehlbauer

From the Instituto Canario de Investigaciones Agrarias,Apartado 60, 38080 La Laguna, Tenerife, Spain (Grajal-Martìn) and USDA-ARS and the Department of Crop andSoil Sciences, Washington State University, Pullman,WA 99164-6434 (Muehlbauer).

Address correspondence to M. J. Grajal-Martìn at the address above.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Resistance to fusarium wilt in peas (Pisumsativum L.) caused by Fusarium oxysporum Schlect. f. sp. pisi race 1 (van Hall) Snyd. & Hans. is conferred by a single dominant gene, Fw. The gene was located in the pea genome by analyzing progenies from crosses involving genetic markers across all pea linkage groups. Phenotyping of the progenies for reaction to race 1 of the fusarium wilt pathogen was determined by field screening in a "wilt-sick" plot in Pullman, Washington. Fw wasshown to be located on linkage group III, about 13 map units from Lap-1 and b and 14 map units from Td. The relatively large distances between these markers and Fw precludes the use of the linked markers in marker-assisted selection for wilt resistance. Additional markers in this region ofthe pea genome will be required if marker-assisted selection for Fw is to be successful.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Fusarium wilt caused by Fusarium oxysporum Schlect. f. sp. pisi race 1 (van Hall) Snyd. & Hans. is a major disease problem of peas (Pisum sativum L.) in most areas.The disease was first found in Wisconsinin the 1920s (Linford 1926) and is now known to have worldwide distribution. A single dominant gene that conferred resistance was found and was designated as Fw (Wade 1929). Most pea cultivars developed since the 1930s have Fw and are resistant to the disease. Resistance is easily identified through selection in field nurseries or by performing inoculation tests of segregating material in controlled environments. Linkage studies (Wade 1929) indicated a loose linkage (greater than 30%) between Fw and Le, a gene controlling internode length. Le was placed on linkage group IV by Lamprecht (1948). The revisions made on the pea linkage map in 1993 and 1996 split linkage group IV into linkage groups IVA and IVB and placed Le and Fw on linkage group IVB (Weeden et al. 1993,1996), while the recently completed consensus map of the Pisum genome tentatively places Fw on linkage group III (Weeden et al. 1998). This latter placement ofFw is based on the results of the presentstudy.

Our objectives were to verify the genetic control of resistance to fusarium wilt race 1, to locate the gene, Fw, that confers resistance in the pea genome relative to other morphological and isozymic markers, and to identify "tags" that could be used to select for wilt resistance in breeding programs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Plant Material
Crosses were made between selected pea lines that contain several morphological and isozymic markers and pea cultivars with known reactions to wilt caused by F. oxysporum f. sp. pisi race 1 (Table 1). Some of the parental lines used were near isogenic for the race 1 resistance gene. For example, we used isolines of William Massey that differed at the Fw locus, with one isoline having the allele for resistance (Fw), while the other the isoline had the allele for susceptibility (fw). Crosses were made either under greenhouse or field conditions and the F1s grown in the greenhouse. Individual F2 plants were grown in the greenhouse and scored for several morphological and isozyme markers. The F2 plants were harvested individually to produce F3 families. Crosses were also made between resistant lines and between susceptible lines and used for tests of allelism.


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  		Table 1.. Crosses made and relevant genetic markers scored in the progeny

 
Disease Reaction
The F3 families were scored for reaction to F. oxysporum f. sp. pisi race 1 in a field disease nursery with a history of predictable race 1 occurrence and where lines susceptible to race 1 do not survive to the flowering stage. Disease reactions were scored when the susceptible controls were completely wilted or dead. The F3 families were scored several times for disease reaction during the growing season in order to avoid escapes and to verify the scoring. We scored the F3 families because the data could be used to infer the genotype of the individual F2 parental plants. F3 families that had some healthy and some wilted pea plants were classified as progenies of F2 plants that were heterozygous for the resistance gene (Fw/fw), F3 families that were uniformly resistant were classified as progenies from F2 plants that were homozygous (Fw/Fw), and uniformly susceptible F3 families were classified as progenies of F2 plants that were homozygous for susceptibility (fw/fw).

Enzyme Assays and Electrophoresis
One or two young pea leaflets of individual F2 plants were ground in 50 µl of 0.1 MTris-HCl (pH 7.5) extraction buffer to produce enzyme extracts (Soltis et al. 1983).These enzyme extracts were absorbed onto filter paper wicks that were placed in 12% starch gels and run in an electric field to separate the isozymes. Different starch gels and electrode buffer systems were used depending on the isozymes to be assayed. Histidine pH 6.1 gels and electrodebuffers (Cardy et al. 1980) were used to resolve 6-phosphogluconate dehydrogenase (PGD, EC 1.1.1.44) and fructose-biphosphate aldolase (ALDO, EC 4.1.2.13). Tris-citrate/lithium borate pH 8.1 gels (Selander et al. 1971) were used to resolve aspartate aminotransferase (AAT, EC2.6.1.1), leucine aminopeptidase (LAP, EC3.4.11.1), phosphoglucomutase (PGM, EC5.4.2.2), and shikimate dehydrogenase (SKDH, EC 1.1.1.25). Citrate/N- (3-aminopropyl)-morpholine pH 6.1 gels and electrode buffers (Clayton and Tretiak 1972) were used to resolve acid phosphatase (ACP, EC 3.1.3.2), esterases, also called arylesterases (EST, EC 3.1.1.2), and methylumbelliferyl ß-D-galactosidases (ß-GAL,EC 3.2.1.23).

Enzyme Nomenclature
Isozymes are identified by an acronym in capital letters followed by a number that distinguishes the different isozymes of an enzyme according to their mobility in starch gels. The most anodal isozyme waslabeled as 1, slower-moving isozymes as 2,and so on (e.g., LAP-1, LAP-2). Allozymes of an isozyme were designated as either fast or slow, with the most anodal designated as fast and the slower allozyme asslow (e.g., LAP-1F, LAP-1S). The most common synonyms to the different isozymes are also provided to facilitate identification.

Data Analysis
Linkage analyses between different markers and Fw were performed using the computer program LINKAGE-1 which employs the method of maximum likelihood (Suiter et al. 1983). We used the pea map developed by Weeden et al. (1996) as a reference for the locations of the different peagenetic markers used in this study.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Susceptible pea plants wilted from the base of the plant upward to the top and died very early in development and before flowering or at the beginning of flowering. Complete wilting of susceptible pea plants took place approximately 45 days after planting in the field season of 1992 and about 60 days after planting in the field season of 1991. Hot and dry weather conditions appeared to be responsible for the early development of disease symptoms in 1992.

Pea plants with long internodes took longer to develop wilt symptoms compared to those with shorter internodes. This observation may be important with regard to tests for resistance to race 1 inorder that enough time be allowed for all susceptible plants to develop clear symptoms.

Progenies from individual F2 plants (F3 families) of each of the crosses, exceptcross 4, fit a 1 uniformly resistant:2 segregating:1 uniformly susceptible ratio. The segregating F3 families inferred an F2 parent population that approximated a 3 resistant:1 susceptible ratio, indicating that a single dominant gene conferred resistance to race 1 of fusarium wilt (Table 2). Tests of allelism indicated that the resistant parents used in this study all carried the same gene for resistance. The F3 families from resistant x resistant crosses were uniformly resistant, while the F3 families from the susceptible x susceptible crosses were uniformly susceptible (data not shown). The dominant Fw gene conferred 100% resistance to the pathogen, and no intermediate disease reaction was observed in any of the crosses.


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  		Table 2.. Reaction of F3 families to Fusarium oxysporum f. sp. pisi race 1 where linkage between Fw and other markers was found

 
Joint segregation of markers used in thisstudy indicated linkage between Fw, Td, b, and Lap-1 (Table 3). Td is a gene for leaf dentation, while b, in the presence of A,confers pink flowers. These linkages were consistent in seven crosses that segregated concurrently for Fw and two or more markers, although the recombination values differed slightly among the crosses. Crosses 1 and 4 segregated for A, the major gene for anthocyanin pigmentation of stems, flowers, pods, and seeds, as well as b. Since A is epistatic to b, only thoseplants classified as A-, about three-fourthsof the population, could be scored for b.


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  		Table 3.. Linkage of Fw with other genetic markers

 
Because slightly different recombination values were found in the different crosses that segregated for the same markers, we tested for homogeneity of the data among the different crosses using the chi-square test. No significant differences among crosses were found for the distribution of data for Fw and b or for the distribution of data for Fw and Lap-1 (Table 3). The grouped data from the different crosses indicated 14% recombination between Tdand Fw, and 13% recombination between b and Fw and between Lap-1 and Fw (Figure 1).



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  		Figure 1.. Putative location of Fw on linkage group III.

 
We did not find linkages between Fw and the other markers used in this study. Wade (1929) reported linkage between Le and Fw, and Lamprecht (1948), believing that Le was on his linkage group 4, placed Fw on that linkage group. Since that time the placement of Fw has not been tested, despite considerable changes to the pealinkage map. Our data indicate that Fw islinked to Lap-1, b, and Td which are located on linkage group III (Weeden et al. 1998). Our results also support the placement of Td on linkage group III. Linkage of Fw with Le, as reported by Wade (1929) and Wells et al. (1948), was not found in this study.

Based on our results, the gene for resistance to F. oxysporum f. sp. pisi race 1 inpeas is placed on linkage group III and linked to Td, b, and the isozymic marker, Lap-1. The location of Fw on linkage group IVB, as previously reported, could not be confirmed. We are now attempting toplace additional polymerase chain reaction (PCR)-based markers on linkage group III in the vicinity of Fw to more precisely determine the gene order and to identify markers that can be used in marker-assisted selection.


   Acknowledgments
 
M. J. Grajal-Martìn wishes to thank the Instituto Nacional de Investigación y Tecnologìa Agraria y Alimentaria (INIA) of Spain forproviding a fellowship during the time this researchwas conducted.


   Footnotes
 
Corresponding Editor: Reid G. Palmer

Received August 20, 2001
Accepted May 17, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

    Cardy BJ, Stuber CW, and Goodman MM, 1980. Techniques for starch gel electrophoresis of enzymes from maize (Zea mays L.). Mimeo series 1317. Raleigh: North Carolina State University.

    Clayton JW and Tretiak DN, 1972. Amine-citrate buffers for pH control in starch gel electrophoresis. J Fish Res Board Can 29:1169–1172.

    Lamprecht H, 1948. Weitere koppelungsstudien an Pisum sativum, insbesondere im Chromosom II (Ar). Agric Hort Genet 10:51–74.

    Linford MB, 1926. A wilt of peas in Wisconsin. Phytopathology 16:75.

    Selander RK, Smith MH, Yang SY, Johnson WE, and Gentry JB, 1971. Biochemical polymorphism and systematics in the genus Peromyscus I. Variation in the old field mouse (Peromyscus polionotus). University of Texas publication 7103. Austin: University of Texas; 49–90.

    Soltis DE, Haufler CH, Darrow DC, and Gastony GJ, 1983. Starch gel electrophoresis of ferns: a compilation of grinding buffers, and staining schedules. Am Fern J 73:9–27.[CrossRef][ISI]

    Suiter KA, Wendel JF, and Case JS, 1983. LINKAGE-1: a PASCAL computer program for the detection and analysis of genetic linkage. J Hered 74:203–204.[Abstract/Free Full Text]

    Wade BL, 1929. Inheritance of fusarium wilt resistance in canning peas. Bulletin 97. Madison: Wisconsin Agricultural Experiment Station; 1–32.

    Weeden NF, Ellis THN, Timmerman-Vaughan GM, Swiecicki WL, Rozov SM, and Berdnikov VA, 1998. A consensus linkage map for Pisum sativum. Pisum Genet 30:1–4.

    Weeden NF, Swiecicki WL, Ambrose M, and TimmermanGM, 1993. Linkage groups of pea. Pisum Genet 25:4,cover.

    Weeden NF, Swiecicki WL, Timmerman-Vaughan GM, Ellis THN, and Ambrose M, 1996. The current pea linkage map. Pisum Genet 28:1–4.

    Wells DG, Walker JC, and Hare WW, 1948. A study oflinkage between factors of resistance to wilt and near-wilt in garden peas. Phytopathology 39:907–912.


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