Inheritance of RAPD Markers in Melon (Cucumis melo L.) J. E. Staub USDA-ARS Vegetable Crops Research Unit , University of Wisconsin-Madison Department of Horticulture, 1575 Linden Dr. Madison WI 53706 Introduction: Genetic markers have been employed in diversity analysis (2,3,4,7,8) and the construction of maps in melon (1,9). The use of random amplified polymorphic DNA (RAPD) has allowed for discrimination of elite (2,7) and unadapted germplasm (3,4,8). Because of their relative low cost and low technological attributes they have been valuable for diversity analysis (2,7). However, except for the mapping of several RAPD marker loci by Baudracco-Arnas and Pitrat (1996), the genetics RAPD markers in melon has not been widely characterized. This is likely due to the relatively low level of RAPD polymorphisms (~15-20%) in melon. The bands used for diversity analysis are repeatable (4,7), but their genetic attributes have not been characterized. This report details the genetics of RAPD markers assessed in F2 progeny segregating in four melon populations. Materials and Methods: Crosses were made between an experimental inbred line SA 200 (‘Chargyne’) x ‘Top Mark’, WI 998 x ‘Top Mark’, Charentais-1 x AR5, and ‘Top Mark’ x AR5. These crosses were made to produce segregating populations that would be useful in mapping disease resistance and sex expression in melon. SA 200 is a gynoecious Charentais market type received from Clause Seed Company (Bretigny-Sur-Orge Cedex, France), Charentais-1 is a gynoecious Charentais market type inbred line received from Petoseed Company (now associated with Seminis, Woodland Calif., USA), AR5 and “Top Mark’ are disease resistant U.S. Western Shipper market types released by the USDA, ARS, and WI 998 is a gynoecious line released by the USDA, ARS. Each F1 was selfpollinated to produce four F2 populations (i.e., SA 200 x ‘Top Mark’, WI 998 x ‘Top Mark’, Charentais1 x AR5, and ‘Top Mark’ x AR5) segregation analyses. DNA was extracted and subjected to PCR using RAPD primers from Operon (Alameda, Calif., USA) and University of British Columbia (UVBC; designated BC; Vancouver, BC, Canada) and electrophoresis was carried out according to Staub et Cucurbit Genetics Cooperative Report 24: 29-32 (2001) al. (7). Initially, a survey of all parents was made using about 1,500 primers to identify polymorphism specific to the contrasting parents in a particular cross. Primer products were identified by their primer designation (e.g., B12 = Operon primer and BC541 = UVBC primer) (Table 1). When a primer produced more than one product that was useable for analysis, it was given a lower case letter designation from cathodal to anodal migration position (e.g., BC541a). Data were obtained for dominant RAPD loci from F2 families and analyzed by chi-square analysis for conformity to expected 3:1 (df = 1) Mendelian single-factor segregation ratios. Results and Discussion: The primers examined yielded between 25 to 40% polymorphisms (band differences between parents) depending on the cross (data not presented). Potentially useful bands were characterized as having a mobility between 200 to 2,500 bp. Putative loci were then identified as those possessing bands that were reproducible and bright and used for segregation analysis (Table 1). Initially, examination of parents of SA 200 x ‘Top Mark’, WI 998 x ‘Top Mark’, Charentais-1 x AR5, and ‘Top Mark’ x AR5 produced 264, 241, 244, and 251 reproducible band differences between respective parents (putative loci) (data not presented). On average, this represents 17% recovery of potentially useable bands (loci) from the survey of 1,500 primers. After this initial assessment, 41, 27, 90, and 38, bands (loci) segregated in a predictable manner in progeny from SA 200 x ‘Top Mark’, WI 998 x ‘Top Mark’, Charentais-1 x AR5, and ‘Top Mark’ x AR5 matings, respectively (Table 1). This represents, on average, 3.3% recovery of useable loci from the initial survey of 1,500 primers. Given the fact that individuals are sometimes misclassified even when scoring loci that historically have proven to reproducible bright RAPD bands, we are attempting RAPD to SCAR conversion at some 29 Table 1. F2 single factor segregation for RAPD primer productsz in melon (Cucumis melo L.). No. Cross Primer SA 200 x TM M7 U13 AT7a AV11 D16 AB17 AC7a AG15 R11 U10 AV11 Y13 AV1 K4 I4b E8 M7 W3a R19b C13 Y15 AM14a Y13 P6 AE3a AL9 E8 AH2b C20 AB4b AI11 W10c AA14 AG2 B11 AK5b G6 AF7 G8 L1 AJ12 A16 AT2 BC226 O2-1 AF7 AL8-1 E6-1 AH9-1 Z9 AB4-2 WI 998 x TM bp F2 675 831 1275 815 1850 831 450 975 1100 870 1900 610 1375 564 831 1300 650 1000 564 1275 1050 1500 575 1200 1250 1650 675 500 700 1325 600 830 1375 1375 1400 1500 1890 1370 1375 564 750 1600 800 1400 1100 947 820 1100 1050 2050 1300 76 76 76 79 77 78 78 78 78 78 74 74 70 50 71 76 76 75 75 73 71 78 74 79 75 75 76 76 76 45 78 78 74 74 80 78 78 70 79 79 75 92 84 92 88 91 75 86 78 78 93 Chi. Obs Exp 57 57 57 59 58 59 58 58 59 59 55 56 53 37 54 58 56 55 55 56 52 60 54 61 58 58 59 55 55 32 56 56 53 53 63 62 55 49 63 63 60 69 63 69 66 68 56 64 58 59 69 57 57 57 59.25 57.75 58.5 58.5 58.5 58.5 58.5 55.5 55.5 52.5 37.5 53.25 57 57 56.25 56.25 54.75 53.25 58.5 55.5 59.25 56.25 56.25 57 57 57 33.75 58.5 58.5 55.5 55.5 60 58.5 58.5 52.5 59.25 59.25 56.25 69 63 69 66 68.25 56.25 64.5 58.5 58.5 69.75 P sq value> NA NA NA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.04 0.05 0.05 0.05 0.07 0.07 0.07 0.09 0.11 0.11 0.11 0.11 0.15 0.21 0.21 0.23 0.24 0.24 0.25 NA NA NA NA 0.00 0.00 0.00 0.00 0.00 0.01 NA NA NA 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.10 0.10 0.10 0.10 0.10 0.10 0.10 NA NA NA NA 0.95 0.95 0.95 0.95 0.95 0.70 Cucurbit Genetics Cooperative Report 24: 29-32 (2001) No. Cross Primer WI 998 x TM AK5 M7-2 AO8 A11-1 AI8-1 AT7-1 AD12 AB16-1 AJ20 J7 AG13 AI9 J7 AB16 AH3-2 AT7-1 V1-1 Z3a D16 F1 O19a AB4b AT2b AU19 BC299 AU2a BC628 I4 AB8 AT15b H2 C20 D9a E6 U13 AA12 R5a AV4 AB17 R11 S4 W10 AF12b AL9a C10 AU2b AB1 AD12 AM1 AP2 BC541a Charent. x AR5 bp 800 750 1600 960 800 860 1100 1375 800 1600 975 520 700 1400 1700 950 564 700 2200 1100 1200 1350 1000 580 700 1400 830 970 1350 1890 600 1000 800 700 1000 830 1000 2027 1000 1300 1910 575 800 1890 900 700 831 1000 950 1375 2100 Chi. F2 Obs 93 87 84 76 89 87 86 78 92 92 88 93 93 87 87 85 82 92 93 93 93 93 93 93 93 90 90 93 93 91 89 93 93 93 93 93 91 91 44 90 93 93 93 93 92 92 93 93 93 93 93 69 66 64 56 68 64 66 57 71 67 68 72 72 63 63 66 59 69 70 70 70 70 70 70 70 67 68 69 69 69 66 71 71 71 71 71 67 67 32 69 68 68 68 68 71 67 72 72 72 72 72 Exp. 69.75 65.25 63 57 66.75 65.25 64.5 58.5 69 69 66 69.75 69.75 65.25 65.25 63.75 61.5 69 69.75 69.75 69.75 69.75 69.75 69.75 69.75 67.5 67.5 69.75 69.75 68.25 66.75 69.75 69.75 69.75 69.75 69.75 68.25 68.25 33 67.5 69.75 69.75 69.75 69.75 69 69 69.75 69.75 69.75 69.75 69.75 P Sq. value > 0.01 0.01 0.02 0.02 0.02 0.02 0.03 0.04 0.06 0.06 0.06 0.07 0.07 0.08 0.08 0.08 0.10 NA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.04 0.04 0.04 0.04 0.06 0.06 0.07 0.07 0.07 0.07 0.07 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 NA 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 30 No. Cross Primer Charent. x AR5 B11 N11a Y10 AF7b AH20 AJ17 AK3 L15 Y15 AF20c AG4a AT2a BC388 J4 AG10a AL8b L1 Q10 AK5 AV11 B14 F3 AX16 D9b J7c BC654 U8 AE2b AF20a AJ12 AT15a L11 C13 K4 N11b U1 AT2c BC388 O19c AF7a AM19 AL9b AF20b AG4b X17 X19 A17b Z3b AI14 AN1 J7b z bp F2 950 1375 775 831 500 700 500 800 1000 580 1400 1100 1100 831 530 400 800 1580 800 1000 1400 400 1100 750 400 1000 974 550 2300 800 2000 1110 1375 700 1000 1904 780 1000 400 840 1900 600 1375 700 700 1910 900 600 700 800 775 93 93 93 93 93 93 92 93 93 93 93 93 93 93 93 93 91 91 78 89 92 92 92 93 93 93 93 93 93 93 91 92 93 93 93 93 93 93 93 93 89 93 93 93 67 93 87 92 93 93 93 Chi. Obs Exp 67 67 67 67 67 67 66 73 73 73 73 73 73 66 66 66 72 72 62 63 65 73 65 74 74 74 65 65 65 65 73 64 75 75 75 75 75 75 64 64 61 76 77 77 44 62 73 61 78 78 61 69.75 69.75 69.75 69.75 69.75 69.75 69 69.75 69.75 69.75 69.75 69.75 69.75 69.75 69.75 69.75 68.25 68.25 58.5 66.75 69 69 69 69.75 69.75 69.75 69.75 69.75 69.75 69.75 68.25 69 69.75 69.75 69.75 69.75 69.75 69.75 69.75 69.75 66.75 69.75 69.75 69.75 50.25 69.75 65.25 69 69.75 69.75 69.75 P sq value> 0.11 0.11 0.11 0.11 0.11 0.11 0.13 0.15 0.15 0.15 0.15 0.15 0.15 0.20 0.20 0.20 0.21 0.21 0.21 0.21 0.23 0.23 0.23 0.26 0.26 0.26 0.32 0.32 0.32 0.32 0.33 0.36 0.40 0.40 0.40 0.40 0.40 0.40 0.47 0.47 0.50 0.56 0.75 0.75 0.78 0.86 0.92 0.93 0.98 0.98 1.10 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.20 No. Cross Primer Charent. x AR5 AF7c T17a AF12a U7 K4 c E6 Z11 b X16 AM18 AF7 AG15 F1 Z18 BC388 AX19 W7 AX20 BC526 AJ20 AT3 AV11 b AF20 AP2 AV11 a K4 a AQ6 H2 AO18 a BC299 U5 U10 O6 T1 AX6 a W10 Z11 a AD12 E1 L2 AB3 Q10 a Z8 TM x AR5 bp 600 1910 1000 1050 831 960 300 575 831 831 974 2027 831 1090 300 831 900 825 1375 1570 831 1400 1100 960 975 947 825 1800 700 564 835 625 947 575 835 1584 1000 795 1000 835 1800 831 Chi. F2 Obs Exp. 93 78 93 87 43 62 42 38 22 45 45 43 43 76 98 40 42 39 44 44 40 22 43 72 42 45 43 43 40 45 45 42 69 45 74 43 73 76 42 42 73 44 69.75 58.5 69.75 65.25 32.25 46.5 31.5 28.5 16.5 33.75 33.75 32.25 32.25 57 73.5 30 31.5 29.25 33 33 30 16.5 32.5 54 31.5 33.75 32.25 32.25 30 33.75 33.75 31.5 51.15 33.75 55.5 32.25 54.75 57 31.5 31.5 54.75 33 79 49 59 52 32 47 31 29 16 33 33 33 33 56 72 29 33 31 31 35 28 15 30 57 29 31 35 35 33 37 37 28 57 29 50 37 61 50 37 37 45 41 P Sq. value > 1.23 1.54 1.66 2.69 0.00 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.07 0.10 0.12 0.12 0.13 0.13 0.15 0.16 0.19 0.22 0.23 0.23 0.30 0.31 0.31 0.39 0.53 0.67 0.55 0.69 0.71 0.86 0.96 0.96 1.74 1.94 0.20 0.20 0.10 0.10 0.95 0.95 0.95 0.95 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.10 0.10 Products designated as primer and lower case letter (e.g., AC7a) (NA = not applicable). Cucurbit Genetics Cooperative Report 24: 29-32 (2001) 31 of the loci (e.g., M7675, U13831, AT7a1275, A161600, AT2800, BC2261400, 02-11100, and Z3a700). Although this type of conversion has proven difficult in cucumber (6), if success is achieved in melon we will make additional conversions. This will allow for the development of a standard array of SCARs markers, and permit their use in diversity analysis and genetic map construction along with previously published codominant markers (5). A standard marker array and the use of reference accessions from previous studies (e.g., 7) will provide powerful set of tools for diversity analysis. Literature Cited 1. 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. 93:57-64. 2. García, E., M. Jamilena, J.I. Álvarez, T. Arnedo, J.L. Oliver, and R. Lozano. 1998. Genetic relationships among melon breeding lines revealed by RAPD markers and agronomic traits. Theor. Appl. Genet. 96: 878-885. 3. Silberstein, L., I. Kovalski, R.G. Huang, K. Anagnostu, M.M.K. Jahn, & R. PerlTreves. 1999. Molecular variation in melon (Cucumis melo L.) as revealed by RFLP and RAPD markers. Sci. Hort. 79: 101-111. isozyme and random amplified polymorphic DNA data for determining intraspecific variation in Cucumis. Gen. Res. Crop Evol. 44: 257-269. 5. Staub, J.E., V. Meglic, and J. D. McCreight. 1998. Inheritance and linkage relationships of melon (Cucumis melo L.) isozymes. J. Amer. Soc. Hort. Sci. 123:264-272. 6. Horejsi, T., J. Box and J. E. Staub. 1999. Efficiency of RAPD to SCAR marker conversion and their comparative PCR sensitivity in cucumber. J. Amer. Soc. Hort. Sci. 124:128-135. 7. Staub, J.E., Y. Danin-Poleg, G. Fazio, T. Horejsi, N. Reis, and N. Katzir. 2000. Comparison analysis of cultivated melon groups (Cucumis melo L.) using random amplified polymorphic DNA and simple sequence repeat markers. Euphytica 115: 225-241. 8. Stepansky, A., I. Kovalski, and R. PerlTreves. 1999. Intraspecific classification of melons (Cucumis melo L.) in view of their phenotypic and molecular variation. Plant Syst Evol 217: 313-332. 9. Wang, Y.H., C.E. Thomas, and R.A. Dean. 1997. A genetic map of melon (Cucumis melo L.) based on amplified fragment length polymorphism (AFLP) markers. Theor. Appl. Genet. 95:791-798. 4. Staub, J.E., J. Box, V. Meglic, T.F. Horejsi, and J.D. McCreight. 1997. Comparison of Cucurbit Genetics Cooperative Report 24: 29-32 (2001) 32