Simple sequence repeat (SSR) and sequence related amplified polymorphism (SRAP) molecular markers were evaluated for detecting intraspecific variation in 38 commercially important peach and nectarine (Prunus persica) cultivars. Out of the 20 SSR primer pairs 17 were previously developed in sweet cherry and three in peach. The number of putative alleles revealed by SSR primer pairs ranged from one to five showing a low level of genetic variability among these cultivars. The average number of alleles per locus was 2.2. About 76% of cherry primers produced amplification products in peach and nectarine, showing a congeneric relationship within Prunus species. Only nine cultivars out of the 38 cultivars could be uniquely identified by the SSR markers. For SRAP, the number of fragments produced was highly variable, ranging from 10 to 33 with an average of 21.8 per primer combination. Ten primer combinations resulted in 49 polymorphic fragments in this closely related set of peaches and nectarines. Thirty out of the 38 peach and nectarine cultivars were identified by unique SRAP fingerprints. UPGMA Cluster analysis based on the SSR and SRAP polymorphic fragments was performed; the relationships inferred are discussed with reference to the pomological characteristics and pedigree of these cultivars. The results indicated that SSR and SRAP markers can be used to distinguish the genetically very close peach and nectarine cultivars as a complement to traditional pomological studies. However, for fingerprinting, SRAP markers appear to be much more effective, quicker and less expensive to develop than are SSR markers.
Riaz Ahmad, Dan Potter, and Stephen M. Southwick
Mikel R. Stevens, Shawn A. Chrisensen, Ammon B. Marshall, JoLynn J. Stevens, Peter Wenzl, Eric Hunter, Jason Carling, and Andrzej Killian
Recently, a technology known as DArT (diversity array technology) has been developed to increase throughput in marker assisted selection (MAS). DArT utilizes microarray technology as a method to potentially compare thousands of molecular markers in one test to a single DNA sample. We used DArT on two sets of interspecific tomato [Solanum lycopersicum (Fla 7613) × S. pennellii (LA 716 or LA 2963)] segregating populations (BC, F2, and F1). We compared over 300 segregating plants to 3840 random tomato genomic fragments. After the 3840 markers were prepared, it took about 2 weeks of laboratory time to perform the experiments. With experience, this time can be reduced. We identified a total of 654 polymorphic markers usable for developing a DArT tomato genetic map. Depending on the particular cross, 13 to 17 linkage groups were identified (LOD 3) per population. Most recently, the amplified polymorphic DNA (AFLP) technique has been used for rapid genetic mapping of large numbers of anonymous genomic fragments. Besides the additional effort and reagents using AFLPs compared to DArT, a desired AFLP polymorphic band is often difficult to clone and process into a PCR based marker, whereas in DArT all markers are already cloned and immediately available for such experiments. A drawback to DArT is that it requires specialized software and equipment and is technically demanding. However, once the equipment and software are secured, techniques are optimized, and segregating populations developed, marker throughput is increased by orders of magnitude. Although challenging, the application of DArT can dramatically increase MAS throughput, thus facilitating quantitative trait and saturated mapping research.
Jan Tiväng, Jim Nienhuis, and O.S Smith
The sampling method was applied to a data-set generated by RFLP molecular marker analysis, representing 37 Zea maize cultivars. A total of 251 enzyme probe-combinations were used yielding a total of 1,205 scores per genotype. Genetic distance was calculated among all 37 entries from subsets of arbitrary and increasing sample size. Each score entry in the subset was selected at random from all possible scores with replacement following each selection. The variance for genetic distance was calculated among all subsets of equal size for all possible cultivar pairs. The pooled pair variance was plotted and compared to random simulation models. Additional comparisons were made contrasting closely vs. distantly related cultivars.
Haejeen Bang, Sunggil Kim, Daniel I. Leskovar, and Stephen King
Fruit color and carotenoid composition are important traits in watermelon. Watermelon fruit color inheritance has revealed that several genes are involved in color determination. Carotenoids are known to have various functions in plants and animals, such as providing antioxidant activity and other health benefits for humans, and UV protection and pigmentation for plants. Differential gene activity in the carotenoid biosynthetic pathway may result in different color determination of mature fruit. Eight genes encoding enzymes involved in the pathway were isolated and their structures were characterized. While obtaining full-length cDNA of these enzymes, two single-nucleotide polymorphisms were detected in a coding region of lycopene β-cyclase (LCYB). These SNP markers showed cosegregation with red and canary yellow fruit color based on the genotyping of two segregating populations. This will lead to development of a codominant molecular marker for the selection of LCYB allele, which may allow breeders to distinguish between red and canary yellow watermelon fruit colors at the seedling stage.
Alexandra B. Napier, Kevin M. Crosby, and Soon O. Park
Muskmelons (Cucumis melo L.) play an important role in the American diet. Ranked as one of the top 10 most-consumed fruits by the USDA, cantaloupe melons have the highest amount of beta-carotene of all the ranked fruits. Beta-carotene, also called pro-Vitamin A, is an essential nutrient required for eye health, and may have the potential, as an antioxidant to reduce the risks associated with cancer, heart disease, and other illnesses. Breeding melons with increased levels of beta-carotene will benefit consumer health. Research has found phytonutrients are most bioavailable when consumed in their fresh form, rather than as vitamin supplements. The high level of beta-carotene found in some melons has a genotypic component, which may be exploited to breed melons high in beta-carotene. Molecular markers and marker-assisted selection (MAS) can be used to increase the efficacy of the breeding process, while lowering breeding costs. An F2 population was created using `Sunrise', the female parent, containing no beta-carotene crossed with `TAM Uvalde', a high beta-carotene variety. A field population consisting of 115 F2 individuals and a greenhouse population containing 90 F2 individuals were grown. The resulting fruit were screened phenotypically and ranked according to beta-carotene content. Chisquare values fit the previously reported model of a single dominant gene for presence of beta-carotene (orange-flesh) vs. absence (green or white flesh). A continuous distribution of beta-carotene concentrations from high to low suggested quantitative inheritance for this trait. Two eight-plant DNA bulks composed of either high or low beta-carotene F2 individuals were screened for polymorphic molecular markers using the amplified fragment-length polymorphism technique.
Mirko Siragusa, Fabio De Pasquale, Loredana Abbate, and Nicasio Tusa
A collection of 18 accessions of sour orange (Citrus aurantium L.) coming from Sicily and other countries was investigated by two polymerase chain reaction (PCR)-based DNA marker technologies. Ten inter-simple sequence repeat (ISSR) primers and fifteen randomly amplified polymorphic DNA (RAPD) primers were used to identify and to evaluate the genetic variability and relationship of accessions. A total of 111 ISSR and 145 RAPD amplified fragments were used to estimate the Dice's coefficient of similarity for cluster analysis using a unweighted pair-group method using an arithmetic averaging (UPGMA) algorithm. The genetic relationships identified using ISSR and RAPD markers were highly concordant, such that the correlation between ISSR and RAPD genetic distance (GD) estimates was r = 0.93. The ISSR and RAPD analysis of 18 sour orange accessions found a high grade of genetic diversity in foreign accessions, while a low variability was detected in local accessions. Sicilian accessions could be grouped in two distinct clusters, including indistinctly plants from three origin regions. Some markers could be linked to the different growing areas. The ISSR and RAPD molecular reference system seems to be suitable for a fine identification of tightly related plants and the obtained results can form the basis for future setting up of Citrus rootstock genetic improvement projects.
Margaret R. Pooler
Many popular crapemyrtle (Lagerstroemia L.) cultivars grown in the United States are interspecific hybrids between L. indica L. and L. fauriei Koehne. The 22 hybrid cultivars released from the U.S. National Arboretum contain primarily genetic material from L. fauriei PI 237884. Examining the genetic diversity ofL. fauriei specimens in the U.S. is valuable because of the historical and economic significance of the species, the increasing interest it is receiving as a source of new cultivars, and its threatened status in the wild. Our objectives were to examine molecular genetic diversity among L. fauriei accessions using Randomly Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphisms (AFLP) markers. Our results indicate: 1) RAPD and AFLP markers are generally consistent in the genetic relationships that they suggest; 2) the L. fauriei germplasm we examined falls into at least three distinct clusters; and 3) the genetic base of cultivated Lagerstroemia could be broadened significantly by incorporating some of this more diverse L. fauriei germplasm into breeding programs.
Marianna Hagidimitriou, Andreas Katsiotis, George Menexes, Constantinos Pontikis, and Michael Loukas
The aim of the present study was to develop a reliable reference database to discriminate between the major Greek olive (Olea europaea L.) cultivars and reveal their genetic relationships, since Greece is considered a secondary center of diversity. In order to establish genetic relationships among the 26 Greek and eight international cultivars, four amplified fragment length polymorphism (AFLP) primer pairs, 12 randomly amplified polymorphic DNA (RAPD) primers, along with measurements from 10 morphological traits, were used. A total of 576 AFLP and 113 RAPD markers were produced. Genetic similarities, estimated using the Jaccard algorithim, ranged from 0.45 to 0.83 for the AFLP data and 0.27 to 0.87 for the RAPD data. The cophenetic correlation coefficients between the genetic similarities and the unweighted pair group method of arithmetic averages (UPGMA) phenograms were 0.77 for the AFLPs, 0.81 for the RAPDs, and 0.69 for the morphological traits. However, limited clustering similarities among the phenograms derived from the three methods were observed. This was also reflected by the low correlation between the three genetic similarity matrices produced (AFLP and RAPD, r = 0.39; AFLP and morphological traits, r = 0.11; RAPD and morphological traits, r = 0.02). According to the molecular results, olive cultivars are clustered according to fruit size but not according to geographical origin. Three of the cultivars tested, `Vasilicada,' `Throumbolia', and `Lianolia Kerkiras', were found to branch distantly to the others, according to the AFLP results, and can be considered as ancient Greek cultivars.
Rita A. Teutonico, Jiwan P. Palta, and Tom C. Osborn
Identification of the genes involved in freezing tolerance in oilseed Erussica could lead to genetic improvement of winter survival of this crop and other species, as well as provide greater understanding of the basis of cold stress tolerance in plants. We developed a genetic linkage map for B. rapa using restriction fragment length polymorphisms (RFLPs) and identified molecular markers which are linked to genes controlling vernalization requirement and freezing tolerance. We mapped the location of a group of cold-regulated (`cor') genes from Arabidopsis thaliana in this population and determined their association with freezing tolerance and vernalization requirement. We developed genetically fixed, recombinant inbred lines of B. rapa to assay the physiological processes involved in these cold responses. Specifically, we measured the differences in lipid composition of the plasma membranes of acclimated and nonacclimated plants of a subset of this population. We will determine if the genes involved in the physiological responses to cold temperature are also associated with the acquisition of freezing tolerance.
Gino Beltran, Geunhwa Jung, Mark Bassett, and James Nienhuis
The development of a complete linkage map including both morphological and molecular markers is important to understand the genetic relationships among quantitatively and qualitatively inherited traits in common bean. The objective of this study was to identify RAPD markers linked to genes for four morphological traits using bulked segregant analysis and to map the markers to a molecular linkage map previously constructed in common bean. Three segregating populations were evaluated. Two BC2F2 populations with dgs (dark green savoy leaf) and blu (blue flower) induced mutant was developed with a Florida breeding line 7-1404 and 5-593 as the recurrent parent. In addition, a BC3F2 population with the y (yellow wax pod) and the arg (silvery green pod) mutants was developed from the Lamprecht line PI 527858 and 5-593 as the recurrent parent. RAPD markers linked in coupling to the morphological traits were detected to be 4.1 cM, 4.3 cM, 7.3 cM, and 12.3 cM distant from the dgs, blu, y, and arg genes, respectively. The linked RAPD markers were mapped in the molecular linkage map previously constructed using recombinant inbred population of the cross PC-50 × XAN-159. In this linkage map, we observed a linkage between the C locus and blu gene whose location was not previously known. In addition, a linkage between an abaxial leaf pubescent gene and dgs gene was observed. These results indicate that integrating morphological markers and molecular markers can result in a more complete genetic linkage map in common bean.