Cucurbit Genetics Cooperative Report 23:69-70 (article230) 2000
Searching for Molecular Markers Linked to ZYMV Resistance in Squash
Rebecca N. Brown and James R. Myers
Department of Horticulture, Oregon State University, Corvallis, OR 97331
The purpose of this study was to find a molecular marker linked to zucchini yellow mosaic virus resistance from Cucurbita moschata 'Nigerian Local'. 'Nigerian Local' is a major source of virus resistance for both Cucurbita pepo and C., moschata. A linked marker could be very useful to breeders in that it would permit selection for virus resistance without actually inoculating plants with ZYMV. Heredity studies have determined that ZYMV resistance in C. moschata is primarily controlled by a single dominant gene (4,5).
We used two BC1 populations for this study: an interspecific population from the cross between a Sunseeds zucchini inbred and 'Nigerian Local', and an intraspecific cross between 'Waltham Butternut' and 'Nigerian Local'. In both cases the backcross was to the susceptible parent. the 'Waltham Butternut' x 'Nigerian Local' population fit a 1:1 segregation ratio as expected. The zucchini x 'Nigerian Local' fit a 1:1 ratio at the first scoring ten days after inoculation with ZYMV, but over the next several weeks most of the "resistant" plants developed virus symptoms of varying severity. These plants were classed as tolerant.
The marker search was conducted using bulk segregant analysis with both RAPDs and AFLPs. Primers were first screened on the parental lines; those which were polymorphic were then screened on the bulks., Markers which produced bands in the resistant bulks and in 'Nigerian Local' were screened on the individuals which comprised the bulks. For the 'Nigerian Local' x 'Waltham Butternut' population there were two resistant bulks and two susceptible bulks. the zucchini x 'Nigerian Local' population was represented by a resistant bulk, two tolerant bulks, and a susceptible bulk. DNA extractions were done using a previously published protocol (2). RAPDs were screened as described elsewhere (1). The AFLP protocol was one originally designed for meadowfoam (3); the restriction enzymes used were EcoRI and MseI.
Results: The 'Waltham Butternut' x 'Nigerian Local' population was screened with 943 RAPD primers, which yielded 4381 scoreable bands. Fourteen percent of the bands were polymorphic between 'Nigerian Local' and 'Waltham Butternut', with 43.3% of the primers giving at least one polymorphic band. This population was also screened with 14 AFLP primer pairs, which yielded 803 scoreable bands. All of the primer pairs gave at least one band which was polymorphic between the parental lines, with an average of 9.4 polymorphic bands per primer pair. Overall, 16.4% of the AFLP bands were polymorphic between the two parents.
The zucchini x 'Nigerian Local' population was screened with 220 RAPD primers. They yielded 1008 scoreable bands, 45% of which were polymorphic between 'Nigerian Local; and the zucchini. Fifty-five percent of the primers gave at least one polymorphic band. This population was not screened with AFLPs.
None of the RAPD primers or AFLP primer pairs amplified bands which were reliably linked to ZYMV resistance or tolerance. The levels of polymorphism between our parents would seem to be sufficient to identify markers linked to resistance. We are still unsure as to exactly why we were unable to find a marker linked to ZYMV resistance. Other efforts to find RAPD markers linked to introgressed disease resistance genes have produced similar results, including an attempt to find markers linked to ZYMV resistance from Cucurbita ecuadorensis introgressed into Cucurbita maxima (N. Weeden, personal communication). One possibility is that the gene for resistance is located near the telomere of a chromosome; such loci are known to be difficult to tag with RAPD markers (S. Knapp, personal communication). Another possibility is that the resistant and susceptible alleles are very similar in sequence, and thus could not be reliably differentiated by RAPDs, which only have an accuracy of 80-90% (6). The AFLP protocol we used was not optimized for Cucurbita. It is possible that further research might reveal AFLP markers linked to ZYMV resistance. In particular, MseI may not be the best restriction enzyme to use with Cucurbita.
We are currently working on a RAPD-based skeleton map of cucurbita using a yellow squash x 'Nigerian Local' BC1 population. One of the traits being mapped is ZYMV resistance. Preliminary data from the mapping population suggests that using ELISA as well as visual symptoms to differentiate resistant and susceptible plants may be key to finding a marker linked to ZYMV resistance. If the resistance gene is telomeric, it may be that other marker technologies such as simple sequence repeats (SSRs) may be more appropriate than RAPDs.
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