Prunus, which includes peach/nectarine, almond, apricot, cherry, and plum, is a large and economically important genus in the family Rosaceae. The size and long generation time of these tree crops have hampered improvement through classical breeding and long-term selection programs. With the advent of DNA-based molecular diagnostics, an exciting era in germplasm improvement has dawned. Efforts are underway, notably in the United States (e.g., California, Michigan, North Carolina, and South Carolina) and the European Community (e.g., England, France, Italy, and Spain), to apply the tools of molecular mapping and marker-assisted selection to this important genus. The objective of these projects is to develop molecular linkage maps of sufficient marker density to tag phenotypic trait loci of agronomic importance. These include traits controlled by single genes (e.g., flower color, compatibility, flesh color, pest resistance), as well as more-complex, quantitative traits (e.g., cold hardiness, tree architecture, sugar content). An immediate outcome of these mapping efforts has been the development of DNA “fingerprints,” allowing for the discrimination of cultivars—both scion and rootstock. The maps will be used by breeders and molecular biologists to monitor gene introgression from wild species into elite lines, for marker-assisted selection of desired trait combinations, and for map-based cloning of specific genes. The molecular markers used in these mapping projects include RFLPs, RAPDs, and microsatellites. Each has their appropriate applications and advantages depending upon the resources at hand and the project's specific goals.
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.
P.W. Simon, P.A. Roberts, and L.S. Boiteux
Nematodes impart significant damage to carrot production worldwide. Genetic resistance was studied for Meloidogyne javanica, one of the three major nematodes affecting carrots in warmer climates. F2, F3, and backcross families of `Brasilia' × B6274 were evaluated for resistance in inoculated seedlings. Resistance was conditioned by one, or two linked, dominant loci. Molecular markers were also evaluated with bulked segregant analysis. Three RAPD markers and AFLPs were associated with resistance loci.
J.G. Tivang, J. Nienhuis, O.S. Smith, and J.S.C. Smith
The statistical properties associated with molecular markers are important when used to characterize germplasm. Evaluation of these properties are necessary for informed selection of one marker system over another. Five different molecular marker systems, Amplified Fragment Length Polymorphism (AFLPs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Random Amplified Polymorphic DNA (RAPDs), Restriction Fragment Length Polymorphism (RFLPs), and Isozymes were used to evaluate 39 elite corn belt inbreds. Each system was characterized for fragment frequency distribution, and band correlation distribution as a measure of independence. A regression model estimating resolution and rate of information addition was constructed using the sampling variance. All marker systems were evaluated according to this model. The model facilitated genetic relationships among the inbreds to be compared at equivalent performance level among all marker systems. Four performance levels resulted in 10 comparisons. Pairwise test of significance were conducted using t tests where the null-distributions were obtained by the bootstrap procedure. The maker system were ranked, assisting breeders in selecting marker systems for germplasm organization.
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.
Patrick J. Conner, Susan K. Brown, and Norman F. Weeden
Molecular markers (isozyme and DNA) have been used to map apple and have helped to elucidate the inheritance of some morphological traits. In this project random amplified polymorphic DNA (RAPD) and isozyme markers were used to create maps for `Wijcik McIntosh, a columnar (reduced branching) sport of `McIntosh' and NY 75441-67, an advanced selection from the multiple disease resistance breeding program. NY 75441-67 is resistant to scab source of resistance from M. floribunda) and resistant to cedar apple rust. `Wijcik McIntosh' is being used in the breeding program as a source of the dominant gene, Co, for reduced branching, but there is also interest in this genotype because of the tremendous variation in plant form observed in progenies segregating for columnar habit. Some of these form variants may be of greater commercial interest than the parental material. Morphological traits examined in this progeny included plant height, stem diameter, suckering, branching habit, spur production, and internode length. The usefulness of molecular markers to pre-select for components of plant form is being examined. Molecular markers promise to aid our understanding and manipulation of quantitative morphological traits.
J.E. Staub and V. Meglic
Scientific disagreement about criteria for accurate classification of similar, if not seemingly identical, cultivars has led to spirited debate in legal and agricultural communities. The lack of universally acceptable working definitions of functional genetic distance and difference, as well as insufficient data on genetic diversity, has made it difficult to define a legal framework for cultivar discrimination. In order to satisfy the “distinctness” criterion during plant patenting, genetic diversity and difference must be described unequivocally in measurable terms. Moreover, the number of markers or other characteristics needed to identify the “nonobvious” nature of the cultigen will determine the breadth of protection under the patent. Increasingly, patent examiners must interpret novelty and distinctness in terms of molecular as well as gross phenotypic (flower color, plant habit, etc.) information. A description of difference using molecular markers may be more difficult compared to a description of function (i.e., how many markers are required to assign difference). Consequently, the effective use of molecular marker information in the legal community will require scientific agreement on the meaning of genetic distance as it relates to genetic difference.
Julie Villand, James Nienhuis, Paul Skroch, and Jan Tivang
Precise cultivar descriptions are necessary to support Plant Variety Protection and utility applications for patent protection. However, accurate discrimination among cultivars is contingent upon the dependability of the method used to delineate lines. The efficiency and reliability of Amplified Fragment Length Polymorphisms (AFLPs), Random Amplified Polymorphic DNAs (RAPDs), microsatellite polymorphisms, and phenotypic traits were studied in order to determine a method's ability to accurately predict pedigree relationships among a set of 20 California processing tomato cultivars. All molecular marker and phenotypic trait data sets were independently produced using identical cultivar seed sources. Data was reduced to a genetic distance measure and presented as a multidimensional scaling (MDS) plot. Principal component analysis using the scored quantitative phenotypic traits was computed and is compared to molecular marker data results. Experimental error, sampling variance, and independence of scored bands for each molecular marker technique are presented. These estimates should assist breeders to determine a sufficient level of characterization, determine a minimum distance considered to be unique, and defend pedigree relationships.
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.
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.