Sang-Min Chung

Diversity in Free-living Populations of Cucurbita pepo (Cucurbitaceae) as Assessed by Random Amplified Polymorphic DNA

Abstract One area of risk assessment for transgenic crops concerns cross-compatible wild and weedy relatives. In squash (Cucurbita pepo), free-living populations are diverse in their distributions, ecologies, histories, and genetic and phenotypic compositions. As part of the effort to understand this diversity, random amplified polymorphic DNA (RAPD) data were collected from 37 wild or weedy populations and 16 cultivars, which together represented all infraspecific taxa of C. pepo. Twenty-six primers yielded 70 scorable and variable markers. The presence/absence of bands for these markers produced a data matrix which was analyzed using cluster analysis. The analysis confirmed the relationships among infraspecific taxa that had been revealed, in part, in previous genetic analyses (e.g., isozymes, chloroplast DNA restriction-site mutations, inter simple sequence repeats). Also supported were findings of varying degrees of gene flow from cultivars into free-living populations. Some of the RAPD variation in subsp. ovifera var. ozarkana populations was found to be correlated with the distribution of the drainage systems along which these populations are dispersed. Finally, the RAPD results support the idea that transgenic gene flow experiments with free-living populations should consider using representatives from each of the three free-living taxa, as well as from genetically or ecologically distinct populations within these taxa. Communicating Editor: Aaron Liston

Assessing the genetic variation in cultivated tomatoes ( Solanum lycopersicum L.) using genome-wide single nucleotide polymorphisms

Tomato (Solanum lycopersicum L.) is an economically important vegetable crop worldwide. Recently, a high-density single nucleotide polymorphism (SNP) array was developed based on genome-wide SNPs in tomato. In this study, we genotyped a collection of 48 Korean elite tomato varieties (26 fresh market and 22 cultivated cherry) using 7,720 SNPs of this array. Out of 6,652 polymorphic SNPs (86.1%) in the entire collection, there were 6,589 SNPs with < 10% missing data. The number of polymorphic SNPs in the fresh market and cultivated cherry subpopulations were 4,733 (61.3%) and 6,087 (78.8%), respectively. To examine the genetic variation between sub-populations, the SNP genotypes of the Korean tomato germplasm were analyzed along with the previously reported data on SNPs of the 277 Solanaceae Agricultural Coordinated Project (SolCAP) varieties (109 fresh market, 27 cultivated cherry, and 141 processing). Principal component analysis, pairwise Fst, and Nei’s standard genetic distance revealed genetic differentiation between these five sub-populations. Moreover, we validated another division within the Korean cherry varieties using the unweighted pair group mean algorithm (UPGMA). The genetic diversity of each sub-population was estimated based on allelic richness and expected heterozygosity. The fresh market and cultivated cherry sub-populations in the Korean tomato germplasm showed similar levels of genetic diversity as the corresponding SolCAP sub-populations. Visualization of the polymorphic information revealed genomic regions that differed between the two sub-populations in the Korean tomato germplasm. These results suggest that diversifying selection for market niches and environmental adaptation has led to allelic variation in cultivated tomatoes in Korea.

The CmACS -7 gene provides sequence variation for development of DNA markers associated with monoecious sex expression in melon ( Cucumis melo L.)

Most melon (Cucumis melo L.) breeding lines in South Korea display andromonoecious sex expression, which necessitates laborious hand emasculation during F1 hybrid seed production. Thus, there is a need to develop monoecious sex types in elite germplasm to obviate self-pollination. Sex expression is associated with floral ethylene production, which, in monecious melon plants, is associated with the A locus. Our study was conducted to develop molecular markers for selection of monoecious plants based on sequence variation inherent in the CmACS-7 gene [encoding 1-aminocyclopropane-1-carboxylic acid synthase (ACS)] that is associated with ethylene production. Full-length CmACS-7 sequences were cloned from a monoecious (MO23) and an andromonoecious (AM24) line. The alignment of those CmACS-7 sequences revealed a single nucleotide polymorphism (SNP; C170T) in exon 1 and an 18 bp indel in the 3′-untranslated region (UTR) of between MO23 and AM24, which was then used to develop a cleaved amplified polymorphic sequence (CAPS) (EX1-C170T) and a sequence characterized amplified region (SCAR) marker (T1ex), respectively. The sex expression and the T1ex SCAR-based genotype of 442 F2 plants derived from a MO23 × AM24 cross was determined. Monoecy and andromonoecy segregated in a 3:1 ratio in F2 progeny, where the sex type of 429 plants (13 plants not classified) co-segregated with the SCAR marker, demonstrating that sex expression regulated by CmACS-7 is controlled by a single dominant gene and that it confers monoecy in line MO23. Allelic variation in 112 geographically diverse melon lines for CmACS-7 as accessed by CAPS EX1-C170T and SCAR T1ex markers indicated that the: 1) exon 1 of CmACS-7 is highly conserved and the SNP/sex expression association detected is highly predictable making it potentially useful for marker-based selection of monoecious plants, and; 2) 18 bp indel mutation in the 3′-UTR was present in various lengths depending on different monoecious melon germplasm.

Plant Transformation via Pollen Tube-Mediated Gene Transfer

Genetic transformation using foreign genes and the subsequent development of transgenic plants has been employed to develop enhanced elite germplasm. Although some skepticism exits regarding pollen tube-mediated gene transfer (PTT), reports demonstrating improved transformation efficiency with PTT systems are increasing and encouraging and the adoption of increasingly refined pollen-mediated methodologies may lead to species-dependent improvements in breeding. Here, we highlight PTT technology as an alternative to genetic transformation.

Development of DNA Markers for Trehalose Synthesis Genes in Brassica rapa L.

High temperature stress might affect the yield and quality of Chinese cabbage. In order to develop cultivars resistant to high temperature stress, we developed polymorphic DNA markers for trehalose synthesis genes related to abiotic stress resistance. A total of 28 Brassica rapa ESTs homologous to trehalose synthesis genes of Arabidopsis were found from the NCBI database. The polymorphic DNA sequences were searched between Chinese cabbages - Chiifu, which is relatively susceptible to high temperature stress, and Kenshin, which is tolerant to high temperature stress. Among the 28 ESTs, we found 10 ESTs that have either insertion/deletion and/or single nucleotide polymorphism between the two cultivars. Those polymorphic sites were then targeted for the development of 10 PCR based markers. These molecular markers related to trehalose genes could be used not only to test their relationship with abiotic stress resistance in Chinese cabbage, but also the development of abiotic stress resistant cultivars using MAS.

Cultivar-To-Wild Population Introgression in Cucurbita pepo subsp. ovifera

Abstract Cultivar-to-wild population transgene flow occurs and can pose potential socio-ecological risks. In the USA, transgenic commercial squash cultivars [i.e., Cucurbita pepo subspecies ovifera var. ovifera] are cultivated in close proximity to cross-compatible wild squash taxa (i.e., vars. ozarkana and texana) that are native to the Americas. This association provides a unique opportunity for assessing cultivar-to-wild genetic introgression in this species. Thus, experiments were conducted using previously characterized random amplified polymorphic DNA (RAPD) to determine whether gene flow has occurred and whether alleles from putative introgression from cultivar-to-wild population exists in free-living populations. Free-living populations of C. pepo from the Ozark Plateau (southern Missouri, northwestern Arkansas, northeastern Oklahoma), Mississippi, Texas, Illinois, Kentucky, and Louisiana were examined using 21 RAPD primers (23 discriminatory bands). Banding morphotypes were useful for discriminating cultivated and wild populations. It appears that C. pepo populations in southeastern Texas and the Ozark Plateau are relatively homogeneous and did not have band morphotypes from cultivated types. Thus, these populations are most representative of var. texana (Texas) and var. ozarkana (the Ozark Plateau). The genetic compositions of accessions from some free-living populations, particularly in Illinois and Kentucky, suggest that squash, pumpkin, and ornamental gourd farming continue to be sources of genetic contamination for nearby wild populations. Thus, gene flow from transgenic cultivars to wild populations is likely, which, in turn, will lead to changes in the genetic structure of such populations.