Potential Utility of RAPD Markers Linked to Fom 2 Gene in Melon (Cucumis melo L.)

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Cucurbit Genetics Cooperative Report 19:61-62 (article 22) 1996

David W. Wolff and Jianling Zhou
Texas Agricultural Experiment Station, The Texas A & M University System, 2415 East Highway 83, Weslaco, TX 78596 [email protected]

Molecular marker identification and utilization in marker-assisted-selection (MAS) can be a valuable tool for the plant breeder, and has been an active research area the past several years. Recently, two research groups have identified RAPD markers linked to the dominant gene conferring resistance to Fusarium wilt races 0 and 1, Fom 2. Wechter et al. (6) used bulked segregant analysis to identify one primer (596) 2.2 centimorgans from Fom 2 in ‘MR-1’. In the first published molecular-marker map in melon, Baudracco-Arnas and Pitrat (2) found two RAPD primers closely linked to Fom 2 in an F2 population derived from ‘Songwhan Charmi’ (PI 161375) and ‘Vedrantais’. E07 and G17 are flanking markers 1.6 and 4.5 cm, respectively, from the FOM 2 gene.

Screening young plant populations for Fusarium wilt resistance is a fairly simple, straightforward test. Based on this criteria, molecular markers appear to have little practical utility in breeding for resistance. An exception to this would be in the case of selecting for resistant genotypes where the pathogen is not native, and its entry is restricted. In this situation, a marker for Fom 2 would be valuable. In addition, a linked molecular marker would facilitate simultaneous multiple-disease screening that would otherwise be difficult, and could facilitate cloning of the resistance gene.

The objective of this study was to assess the applicability of these markers for use in marker-assisted selection of Fusarium wilt race 1 resistance in melon. The initial question of interest is, are these primers specific to populations derived from the source genotype of the identified primer, or are they usable in other populations? The results may indicate the ancestry and relatedness of Fom 2 genes in different melon genotypes.

Materials and Methods. Young leaves were harvested from greenhouse grown plants and DNA was extracted according to the protocol of Baudracco-Arnas (1). Along with primer source genotypes (‘MR-1’, ‘Vedrantais’) other known susceptible (‘Ananas Yokenum’ [AY]. ‘Topmark’) and resistant genotypes (PI 161375) were screened with the 3 RAPD primers (Table 1). In addition, a subsample from a segregating backcross population (MD 8654 used as the Fom 2 source) was also screened with the primers. The PCR reactions followed the general protocol of Giovanni et al. (3). Reactions (25 μl volumes) were carried out in a Perkin Elmer 480 thermocycler. Each reaction contained 2 μl DNA (5 ng/ μl stock), 17 μl sterile distilled H20, 2.5 μl 10x PCR buffer, 1.2 μl GATC nucleotides, 0.1 μl MgCl2 (100 mM),0.1 μl Perkin-Elmer AmpliTaq polymerase, and 2 μl of the 10-mer primer (3 ng/ μl stock) synthesized by Gibco BRL Life Technologies (Grand Island, New York). The following thermocycles were used: 1 cycle, 94 C 10 min; 44 cycles, 94 C 1 min / 36 C 1 min / 72 C 2 min; 1 cycle: 94 C 1 min / 36 C 1 min / 72 C 10 min. Amplified products were separate don an 2.0% agarose gel run at 5v/cm for 4 hours. Gels were scored for the presence or absence of the linked fragment of each primer: 595 – 1.6 kb, E07 – 1.3 kb, G17 – 1.0 kb.

Results and Discussion. Several gels were run with each primer to evaluate the utility of each for selecting resistant genotypes. Of the 3 primers, we had he most difficulty in producing consistent amplification of the linked fragment with 596. Therefore, no scoring could be done with primer 596. PCR conditions will have to be tested to optimize the reactions. The unstable nature of PCR based RAPDs is well documented, and has been a problem in Cucumis (5).

The other two primers identified in ‘Vedrantais’ did produce the expected fragment profile on most gels. Scoring G17 was difficult because of a fragment in both resistant and susceptible genotypes that was only 50 bp in size smaller than the linked fragment. This fragment (1.0 kb) is linked to the susceptible allele (fom 2) and was seen in the susceptible ‘Vedrantais’ (source) and ‘Ananas Yokneum’, but was absent in the resistant genotypes PI 161375 and ‘MR-1’. In a small sample of lines segregating for Fom 2 from the test popular ion, the susceptible band appeared in some, but not al susceptible genotypes. Primer E07 performed like G17 in the parental lines, and with a subset of segregating lines, did accurately predict phenotype. The linked fragment, however, was not consistently amplified in every gel. E07 is more tightly linked to Fom 2 than G17 (1.6 vs. 4.5 cm), and therefore would more consistently identify the correct phenotype.

Wechter et al. (6) did not find the 1.6 kb fragment from primer 596 in 3 other resistant genotypes tested. Their data indicate that this primer may be utilized only in populations derived from MR-1. We were not able at this time to get consistent data with this primer, and therefore cannot contribute further data on this primer.

Data from the two primers linked to the susceptible allele in ‘Vedrantais’ indicate potential success in identifying susceptible melon individuals from diverse backgrounds. In the small sample thus far tested, these primers have co-segregated with phenotype. Assuming that susceptibility is the older, ancestral form of the Fom 2 gene, a linked marker to the susceptible allele may be usable over a wider array of genotypes than one linked to the resistant allele. This hypothesis can only be tested after evaluating many known susceptible and resistant genotypes with these primers, and determining co-segregation with phenotype.

The fragment from E07 and G17 is linked to the susceptible allele, thus the resistant genotype has no band. This is not desirable when using MAS, since a null band could result from a failed PR reaction, and not the absence of the linked fragment (i.e., a true, resistant plant). Contrarily, the advantage of a linked susceptible marker is that homozygous dominant individuals could be identified from heterozygous individuals. Wechter et al. (6) identified a second primer which was linked to the susceptible allele; however, they did not pursue this further because of the above mentioned problem. It would be interesting to test this primer along with the other two from ‘Vedrantais’.

The problems of RAPD consistency and difficulty in scoring (multiple fragments per single primer) could be overcome if RAPDs are converted to SCAR (sequence characterized amplified regions) markers (4). This would become attractive economically if identified RAPD primers could be utilized across diverse genotypes. The preliminary data presented here shows that two primers linked to the susceptible allele may work across melon genotypes, although more data is needed to confirm this.

Table 1, Characteristics of RAPD primers linked to the dominant, resistant gene conferring resistance to Fusarium wilt races 0 and 1 in melon (Fom 2).

Primer

Sequence 5′ – 3′

Reference

Source

Genotype/phenotype

Fragment size (kb)
596 CC CTC GAA T (6) ‘MR-1’/Resist. 1.6
E07 AGA TGC AGC C (2) ‘Vedrantais’/Susc. 1.3
G17 ACG ACC GAC A (2) ‘Vedrantais’/Susc. 1.0

Literature Cited

  1. Baudracco-Arnas S. 1995. A simple and inexpensive method for DNA extraction from Cucumis melo L. Cucurbit Genetics Coop. Rept. 18:50-51.
  2. Baudracco-Arnas S. and M. Pitrat. 1996. A genetic map of melon (Cucumis melo L.) with RFLP, RAPD, isozyme, disease resistance and morphological markers. Theor. Appl. Genet. (In Press).
  3. Giovannoni, J.J., R.A. Wing, M.W. Ganal and S.D. Tanksley. 1991. Isolation of molecular markers from specific chromosomal intervals using DNA pools from existing mapping populations. Nucl. Acid. Rees. 19:6553-6558.
  4. Paran I. and R.W. Michelmore. 1993. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor. Appl. Genet. 85:985-993.
  5. Staub J., J. Bacher and K. Poetter. 1996. Sources of potential errors in the application of random amplified polymorphic DNAs in cucumber. HortScience 32:262-266.
  6. Wechter W.P., M.P. Whitehead, C.E. Thomas and R.A. Dean. 1995. Identification of an randomly amplified polymorphic DNA marker linked to the Fom 2 Fusarium wilt resistance gene in muskmelon MR-1. Phytopathology 85:1245-1249.