Highly polymorphic microsatellites or simple sequence repeat (SSR), along with sequence characterized amplified region (SCAR) and single nucleotide polymorphisms (SNP), markers are reliable, cost-effective, and amenable for large scale analyses. Molecular polymorhisms are relatively rare in cucumber (Cucumis sativus L.) (3% to 8%). Therefore, experiments were designed to develop SSR, SCAR and SNP markers, and optimize reaction conditions for PCR. A set of 110 SSR markers was constructed using a unique, strategically applied methodology that included the GeneTrapper (Life Technologies, Gaithersburg, Md.) kit to select plasmids harboring microsatellites. Of these markers, 58 (52%) contained dinucleotide repeats (CT, CA, TA), 21 (19%) possessed trinucleotide repeats (CTT, ATT, ACC, GCA), 3 (2.7%) contained tetranucleotide repeats (TGCG, TTAA, TAAA), 4 (3.6%) enclosed pentanucleotide repeat (ATTTT, GTTTT, GGGTC, AGCCC), 3 (2.7%) contained hexanucleotide repeats (CCCAAA, TAAAAA, GCTGGC) and 21 possessed composite repeats. Four SCARs (L18-3 SCAR, AT1-2 SCAR, N6-A SCAR, and N6-B SCAR) and two PCR markers based on SNPs (L18-2H19 A and B) that are tightly linked to multiple lateral branching (i.e., a yield component) were also developed. The SNP markers were developed from otherwise monomorphic SCAR markers, producing genetically variable amplicons. The markers L18-3 SCAR and AT1-2 SCAR were codominant. A three-primer strategy was devised to develop a codominant SCAR from a sequence containing a transposable element, and a new codominant SCAR product was detected by annealing temperature gradient (ATG) PCR. The use of a marker among laboratories can be enhanced by methodological optimization of the PCR. The utility of the primers developed was optimized by ATG-PCR to increase reliability and facilitate technology transfer. This array of markers substantially increases the pool of genetic markers available for genetic investigation in Cucumis.
Gennaro Fazio, Jack E. Staub and Sang Min Chung
Sang-Min Chung, Jack E. Staub and Gennaro Fazio
Chilling temperatures (≤12°C) can cause substantial economic damage to cucumber (Cucumis sativus L.) plants. Previous studies suggest chilling tolerance trait is controlled by nuclear gene(s). To investigate inheritance of chilling injury in cucumber, cucumber lines [susceptible GY14 (P1), tolerant `Chipper' (P2), and tolerant `Little John' (P3)], and their exact reciprocal F1 and F2 cross-progeny were evaluated to determine the inheritance of chilling injury at the first true-leaf stage when challenged at 4 °C for 5.5 hours. The mean chilling ratings [1(trace) to 9(dead)] of progeny comparisons were F1(P1 × P2) = 6.2 vs. F1(P2 × P1) = 1.6; F2(P1 × P2) = 6.4 vs. F2(P2 × P1) = 2.7; F1(P1 × P3) = 5.4 vs. F1(P3 × P1) = 1.7; and F2(P1 × P3) = 5.8 vs. F2(P3 × P1) = 2.2. These data suggest that chilling tolerance was maternally inherited as is the chloroplast genome in cucumber. Parents, reciprocal F1, and F2 progeny were evaluated for variation using random amplified polymorphism DNA (RAPD). Although no maternally inherited RAPD markers were detected, polymorphic and paternally inherited RAPD bands AD21249, AV8916, and AV8969 amplified by AD2 and AV8 primers were cloned and sequenced. A BLAST search of these sequences suggested that their origin is likely cucumber mitochondrial DNA. These results indicate that the mitochondria genome is not associated with the chilling tolerant trait because this genome is paternally inherited in progeny derived from this reciprocal mating. Therefore, the results of maternally inherited chilling tolerant trait and paternally transmitted mitochondria genome support that the chilling tolerant trait as identified is likely associated with the chloroplast genome which is maternally transmitted in cucumber.
Jack E. Staub, Zhanyong Sun, Sang-Min Chung and Richard L. Lower
Cucumber (Cucumis sativus L. var. sativus; 2n = 2x = 14), has a narrow genetic base (3% to 8% polymorphism). Nevertheless, several genetic maps exist for this species. It is important to know the degree of colinearity among these maps. Thus, the positions of random amplified polymorphic DNAs, sequenced characterized amplified regions, simple sequence repeat, restriction fragment length polymorphisms, and fluorescent amplified fragment length polymorphism markers were compared in four maps. A previously unreported map was constructed in a narrow cross (processing line 2A × Gy8; C. s. var. sativus; ≈7% polymorphism) and compared with the three published maps [two narrow-based (processing type; C. s. var. sativus; 8% to 12% polymorphism) and a broad-based (C. s. var. sativus × C. s. var. hardwickii (R.) Alef. ≈12%)]. Common makers were identified in seven linkage groups, providing evidence for microsynteny. These common markers were used as anchor markers for map position comparisons of yield component quantitative trait loci. The relative order of anchor markers in each of six linkage groups (linkage groups 1, 2, and 4–7) that had two or more anchor markers within each group was colinear, and instances of microsynteny were detected. Commonalities in the position of some yield component quantitative trait loci exist in linkage groups 1 and 4 of the maps examined, and the general synteny among these maps indicates that identification and mapping of additional anchor markers would lead to successful map merging to increase cucumber map saturation for use in cucumber breeding.
James D. McCreight, Jack E. Staub, Anabel López-Sesé and Sang-Min Chung
Genetic variation among 378 melon (Cucumis melo L.) germplasm accessions collected in India in 1992 and 26 accessions in China in 1994 was evaluated with 19 isozyme loci. `Top Mark' and `Green Flesh Honeydew', which represented two distinct C. melo ssp. melo L. groups, Cantalupensis and Inodorus, respectively, were used as reference cultivars. Genetic distances among accessions were calculated, and an initial cluster analysis using these distances resulted in 148 groups of varying size, ranging from two to 47 accessions. One accession from each of the 148 groups was chosen at random and used in a second cluster analysis that identified 11 accession groups. Group 1 was unique and consisted of only two C. melo ssp. agrestis (Naudin) Pangalo accessions. Two large branches were detected at cluster node 2. One branch was comprised of three groups of 3, 12, and 34 accessions, while the other branch contained seven groups of 2, 3, 14, 16, and 47 accessions, and the reference cultivars. Of the 148 accessions, 132 were from 41 sites in Rajasthan and Madhya Pradesh, India, which were distributed unequally across the 11 groups. The 14 Chinese accessions originating from seven provinces were also dispersed unequally in the four major cluster groups. `Top Mark' and `Green Flesh Honeydew' were genetically distinct and uniquely clustered in the same group. These results indicate that additional collections of melon germplasm should be made in eastern and southern India.