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David Byrne

Fruit and ornamental breeders were surveyed about their use of molecular markers in either their breeding programs or in their related research programs. Responses were obtained from over 100 fruit and ornamental breeding programs from throughout the world. Of these, less than 50% used molecular markers in their programs. The two most common uses of these markers were for studies in plant identification and diversity. These were followed by the use of markers in developing molecular maps, in discovering molecular tags and/or trying to identify the genes for specific plant traits, for marker assisted selection, and finally, for the elucidation of plant taxonomy. In conclusion, although there is much research in this area, few programs are actually using markers in the context of an applied breeding program. The major reason for this situation is the lack of available markers and the cost of using these markers to screen large numbers of progeny. Those that use markers in their breeding tend to use them to verify the genotype of the parents or confirm the genotype of selected seedlings rather than screen unselected seedlings.

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Eric T. Stafne, John R. Clark, and Kim S. Lewers

A tetraploid blackberry population that segregates for two important morphological traits, thornlessness and primocane fruiting, was tested with molecular marker analysis. Both randomly amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to screen a population of 98 genotypes within the population plus the two parents, `Arapaho' and `Prime-Jim' (APF-12). RAPD analysis averaged 3.4 markers per primer, whereas SSR analysis yielded 3.0 markers per primer pair. Similarity coefficient derived from the Dice index averaged over all individuals was 63% for RAPD markers, 73% for SSR markers, and 66% for RAPD and SSR markers together. The average similarity coefficients ranged from a high of 72% to a low of 38% for RAPD markers, 80% to 57% for SSR markers, and 73% to 55% for both. Comparison of the parents indicated a similarity of 67% for RAPD markers, 62% for SSR markers, and 67% for both. This is similar to a previous study that reported the similarity coefficient at 66%. Although inbreeding exists within the population, the level of heterozygosity is high. Also, evidence of tetrasomic inheritance was uncovered within the molecular marker analysis. This population will be used to identify potential markers linked to both morphological traits of interest. Further genetic linkage analysis and mapping is needed to identify any putative markers.

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Luisa Monte-Corvo, Luis Goulão, and Cristina Oliveira

Inter-simple sequence repeat (ISSR) markers were used for cultivar identification and for determination of the phenetic relationships among 24 pear cultivars (Pyrus communis L.). The ability of several molecular marker systems including randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP), inter-simple sequence repeats (ISSR), simple sequence repeats (SSR), and selective amplification of microsatellite polymorphic loci (SAMPL) to detect variation among clones of the most significant Portuguese cultivar, Rocha, was also investigated. Each of the eight ISSR primers tested was able to distinguish the 24 pear cultivars. The ISSR primers generated 337 markers, 79.5% of which were polymorphic. The cultivar dendrogram obtained with the ISSR marker data was very similar to that obtained with previous RAPD+AFLP analysis, confirming the genetic divergence of `Pérola', `Carvalhal' and `Lawson' from the other cultivars. Eight out of 15 apple [Malus sylvestris (L.) Mill. var domestica (Borkh.) Mansf.] SSR primers tested also amplified microsatellites in pear. None of the five molecular marker systems analyzed (with a total of 1082 markers) detected reproducible polymorphisms among the nine `Rocha' clones, in spite of the presence of clear phenotypic differences.

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Patrick J. Conner, Susan K. Brown, and Norman F. Weeden

Two half-sib populations (cross 1 = `Wijcik McIntosh' (WM) × NY 75441-67, and cross 2 = WM × NY 75441-58) were used to create maps for the parents and to find RAPD or isozyme markers for qualitative and quantitative traits. WM is a sport of `McIntosh' and is heterozygous for the dominant columnar (Co) gene for reduced branching. WM is of great interest in breeding because of the tremendous effect of the Co gene on many aspects of plant form. NY 75441-67 and NY 75441-58 are advanced selections with commercial fruit quality and resistance to scab (Vf resistance from M. floribunda). Traits examined included both tree (plant height, stem diameter, suckering, branching habit, leaf break, burr knot production) and fruit (size, shape, color, stem length, seed number) characters and fruit quality traits (pH, acid content, Brix). The conservation of RAPD markers in these closely related crosses will be examined and the usefulness of molecular markers to preselect for components of plant form and fruit quality will be discussed. Molecular markers will increase the efficiency of the apple breeding program by aiding the understanding and manipulation of complex genetic traits.

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Mwamburi Mcharo*, Don Labonte, Chris Clark, and Mary Hoy

Using two sweetpotato (Ipomoea batatas (L.) Lam) F1 populations from diverse environments we investigated the AFLP marker profiles of the genotypes for association studies between the molecular markers and southern root-knot nematode (Meloidogyne incognita) resistance expression. Population one consisted of 51 half-sib genotypes developed at the Louisiana State Univ. AgCenter. The second population consisted of 51 full-sibs developed by the East African and International Potato Center sweetpotato breeding programs. Results for nematode resistance expression indicate a binomial distribution among the genotypes. Using analysis of molecular variance, logistic regression and discriminant analysis, AFLP markers that are most influential with respect to the phenotypic trait expression were selected for both populations. A comparative analysis of the power of models from the two statistical models for southern root-knot nematode resistance class prediction was also done. The diversity and possible universal similarity of influential markers between the two populations and the expected impact in sweetpotato breeding programs will be discussed.

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James D. Kell

The use of RAPD markers in disease resistance breeding has been successfully demonstrated in horticultural crops. The identification of RAPD markers and their potential role in disease resistance breeding was first demonstrated in tomato; the procedure of bulk segregant analysis for detecting linkages between RAPD markers and genes conditioning resistance to downy mildew was described in lettuce; the use of linked RAPD markers to facilitate the efficient pyramiding of epistatic rust resistance genes to prolong their durability has been illustrated in common bean; the development of allele specific or sequence characterized primers linked to resistance genes has been achieved in pea, bean, tomato, and lettuce Indirect selection for disease resistance based on molecular markers is not a replacement for classical breeding procedures. Used as tools, markers offer breeders unique opportunities to combine resistance to an array of different pathogens and efficiently pyramid epistatic resistance genes for highly variable fungal pathogens. This would not always be possible in the absence of linked markers.

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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.

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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.

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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.

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Matthew D. Robbins, Mikel R. Stevens, Gennaro Fazio, and Gennaro Fazio

Fusarium crown and root rot (crown rot) develops on tomato from the fungus Fusarium oxysporum f.sp. radicis-lycopersici (FORL). Genetic resistance to crown rot was previously introduced into the cultivated tomato from the wild species Lycopersicon peruvianum and found to be a single dominant gene, Frl, on the long arm near the centromere of chromosome 9 of the tomato genome. In an effort to identify molecular markers tightly linked to the gene, Ohio 89-1 Fla 7226, Fla 7464, `Mocis', and `Mopèrou', lines homozygous for Frl (resistant), were screened with restriction fragment length polymorphism (RFLP) markers in comparison to Fla 7482B and `Monalbo', lines homozygous for Frl + (susceptible). Frl was determined to be between the RFLP markers CT208 and CD8. These two markers are separated by a genetic map distance of 0.9 cM according to Pillen et al. (1996). In addition, we screened a pool of eight resistant plants against a pool of nine susceptibles from a BC1 population segregating for Frl for amplified fragment length polymorphism (AFLP) markers. Fazio et al. (1998) previously determined that crossover events occurred in these 17 plants between Frl and a rapid amplified polymorphic DNA (RAPD) marker, UBC194. Our research has indicated that UBC194 is also between CT208 and CD8 on the centromeric side of Frl. Of the 62 AFLP primer combinations tested, 34 showed more than 63 strong polymorphisms in linkage to resistant phenotypes.