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Majid R. Foolad

In tomato, Lycopersi conesculentum Mill., currently there are >285 known morphological, physiological and disease resistance markers, 36 isozymes, and >1000 RFLPs, which have been mapped onto the 12 tomato chromosomes. In addition, currently there are >162,000 ESTs, of which ∼3.2% have been mapped. Several tomato genetic maps have been developed, mainly based on interspecific crosses between the cultivated tomato and its related wild species. The markers and maps have been used to locate and tag genes or QTLs for disease resistance and other horticultural characteristics. Such information can be used for various purposes, including marker-assisted selection (MAS) and map-based cloning of desirable genes or QTLs. Many seed companies have adopted using MAS for manipulating genes for a few simple morphological characteristics and several vertical disease resistance traits in tomato. However, MAS is not yet a routine procedure in seed companies for manipulating QTLs although it has been tried for a few complex disease resistance and fruit quality characteristics. In comparison, the use of MAS is less common in public tomato breeding programs, although attempts have been made to transfer QTLs for resistances to a few complex diseases. The potential benefits of marker deployment to plant breeding are undisputed, in particular for pyramiding disease resistance genes. It is expected that in the near future MAS will be routine in many breeding programs, taking advantage of high-resolution markers such as SNPs. For quantitative traits, QTLs must be sought for components of genetic variation before they are applicable to marker-assisted breeding. However, MAS will not be a “silver bullet” solution to every breeding problem or for every crop species.

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Hong Y. Yang, Schuyler S. Korban, Jutta Kruger, and Hanna Schmidt

Apple scab, caused by Venturia inaequalis (Cke.) Wint., is the most serious disease of apple trees. Resistance to V. inaequalis, derived from the small-fruited species Malus floribunda 821, is determined by a major dominant gene Vf. Our major objective is to identify RAPD markers linked to the Vf gene. The approach in this paper is based on the introgression of the Vf gene from M. floribunda into commercial cultivars. Almost 200 random sequence decamer-primers have been used to screen a pair of bulked samples and the donor parent M. floribunda clone 821 for markers linked to the Vf gene conferring resistance to apple scab. A single primer has been identified which generated a PCR fragment, OPK16/1300, from the donor parent M. floribunda clone 821 and the scab-resistant selections/cultivars bulk, but not from the scab-susceptible recurrent parent bulk. Co-segregation analysis using a segregating apple progeny and polymorphism analysis of individual scab-resistant Coop selections/cultivars have confirmed that this marker is linked to the scab-resistance gene Vf. OPK16/1300 has since been cloned and sequenced. Sequence-specific primers of 25 oligonucleotides based on the marker have been synthesized and used to screen further M. floribunda clone 821, scab-susceptible apple cultivars, scab-resistant apple cultivars, and scab-resistant Coop selections. The sequence-specific primers have identified polymorphisms of OPK16/1300 based on the presence or absence of a single band.

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Robert R. Krueger and Mikeal L. Roose

New potential citrus germplasm accessions may be received as seed rather than budwood, thereby reducing phytosanitary risks. However, trueness-to-type may be an issue with seed materials because many varieties produce both apomictic (nucellar) and sexual (zygotic) embryos and most citrus is fairly heterozygous. To identify nucellar seedlings of polyembryonic types and to retain these as representing the type, we screened 1340 seedlings from 88 seed sources for markers amplified with two inter-simple sequence repeat (ISSR) primers. Sixteen seed sources produced no seedlings classified as being of nucellar origin. Among the remaining seed sources, seedlings classed as nucellar were identified for potential addition to the collection. In 37 accessions, both nucellar and zygotic seedlings were detected, and in some cases both types were retained. Inclusion of established accessions of the same cultivar group in the analysis allowed an initial assessment of similarity to existing accessions. This technique improved the efficiency of acquiring new germplasm of polyembryonic types by seed. The method identifies those seed sources that produce few or no nucellar seedlings, but it is not useful for determining which seedlings of monoembryonic types should be retained in collections.

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M. Dolores Loureiro, M. Carmen Martínez, Jean-Michel Boursiquot, and Patrice This

`Albariño' (Vitis vinifera L.) is an important grape cultivar in Spain, morphologically diverse but subject to much misnaming. The objectives of the present work were to correct some of the more common misnamings concerning `Albariño' and to evaluate the genetic variability within this cultivar by analyzing DNA polymorphisms using randomly amplified polymorphic DNA (RAPD) markers and microsatellite techniques. Several accessions of `Albariño' (16 accessions from Misión Biológica de Galicia, one accession from El Encin, one accession from Rancho de la Merced), related cultivars (`Alvarinho', `Caíño blanco', `Cainho branco', `Loureiro'), and cultivars presumably identical to misnomers (`Savagnin blanc' and `Gewürztraminer') were analyzed using 20 RAPD markers and six microsatellite loci. Both techniques revealed polymorphism among `Albariño', `Caíño blanco', `Albariño' from Rancho de la Merced and `Loureiro'. No polymorphism was detected among the 16 `Albariño' accessions from Galicia, the `Albariño' accession from El Encin and `Alvarinho', nor among the `Albariño' accession from Rancho de la Merced, `Savagnin blanc' and `Gewürztraminer', nor between `Caíño blanco' and `Cainho branco'. These results enabled us to clarify the main misnomers concerning these cultivars. The absence of polymorphism among the true `Albariño' accessions did not allow the detection of any clonal variation. The suitability of both techniques for defining the cultivar level for grapevine is discussed.

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Zhanao Deng, Jinguo Hu, Fahrettin Goktepe, Brady A. Vick, and Brent K. Harbaugh

Cultivated caladiums are valued for their bright colorful leaves and are widely used in containers and landscapes. More than 1500 named cultivars have been introduced during the past 150 years, yet currently only about 100 cultivars are in commercial propagation in Florida. Caladium tubers produced in Florida account for 95% of the world supplies. Loss of caladium germplasm or genetic diversity has been a concern to future improvement of this plant. In addition, the relationship among the available cultivars, particularly those of close resemblance, has been lacking. This study was conducted to assess the genetic variability and relationship in commercial cultivars and species accessions. Fifty-seven major cultivars and 15 caladium species accessions were analyzed using the target region amplification polymorphism marker technique. This marker system does not involve DNA restriction or adaptor linking, but shares the same high throughput and reliability with the amplified fragment length polymorphism system (AFLP). Eight primer combinations amplified 379 scorable DNA fragments among the caladium samples. A high level of polymorphism was detected among the species accessions as well as among cultivars. These markers allowed differentiation of all the cultivars tested, including those hardly distinguishable morphologically. Clustering analysis based on these DNA fingerprints separated the cultivars into five clusters and Caladium lindenii far from other caladium species. The availability of this information will be very valuable for identifying and maintaining the core germplasm resources and will aid in selecting breeding parents for further improvement.

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Martha Dávila, Dermot Coyne, Shree Singh, and Guenhwa Jung

The genes involved in F1 seedling abnormal development and lethality in inter-gene pool crosses have been designated as Dl1 (MesoAmerican=MA) and Dl2 (Andean=A) (Shii et al., 1980, J. Hered. 71:218–222). The different degrees of leaf crippling (C) in segregating populations of crosses was due to the interaction between the Dl1 or Dl2 loci, growing environment, and the lcr allele (Singh and Molina, 1996, J. Hered., In press). The objective was to identify RAPD markers linked to the genes for crippling (lcr) and seedling lethality (Dl) using the bulked segregation analysis procedure for F2 of MA × A crosses. Crosses were made between C lines, FB 10413-24-2, WA 7807-305, and TY 5578-220 and normal (N) parents and tester stocks for Dl1 and Dl2 genes. The F2 FB 10413-24-2 × Carioca segregated 13 N:3C. F3 families segregated 3N:1C. RAPD marker OPB-10 was linked to Lcr at 31.2 cM. F3 families segregated 1N:3C. RAPD marker OPO16 was linked to Dl1 at 27 cM. The F2 WA-7807-305 × Rio Tibagi segregated 3N:1C. RAPD marker OPS-03 was linked to Lcr at 32.6 cM.

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Hong Y. Yang, Schuyler S. Korban, Jutta Krüger, and Hanna Schmidt

Almost 200 random sequence decamer primers were used to screen a pair of bulked samples of apple (Malus ×domestica Borkh.) DNA and that of the donor parent Malus floribunda Sieb. clone 821 for molecular markers linked to the Vf gene conferring resistance to apple scab [Venturia inaequalis (Cke.) Wint.]. Identified was a single primer that generated a polymerase chain-reaction (PCR) fragment, OPAR4/1400, from the donor parent M. floribunda clone 821 and the scab-resistant selections/cultivars bulk, but not from the scab-susceptible recurrent-parent bulk. Cosegregation analysis using a segregating apple progeny and polymorphism analysis of individual scab-resistant selections/cultivars confirmed that this marker was linked to the scab-resistance gene Vf OPAR4/1400 was then cloned and sequenced. Sequence-specific primers of 25 oligonucleotides based on the marker were developed and used to screen further M. floribunda clone 821, scab-susceptible apple cultivars, scab-resistant apple cultivars, and scab-resistant Purdue, Rutgers, and Univ. of Illinois apple breeding program selections. The sequence-specific primers identified polymorphisms of OPAR4/1400 based on the presence or absence of a single band. This molecular marker is at a distance of about 3.6 cM from the Vf gene.

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Mohammed A.M. Aly, Robert G. Fjellstrom, Gale H. McGranahan, and Dan E. Parfitt

Somatic embryos derived from walnut (Juglans regia L.) ovule tissues were evaluated to determine whether they were of zygotic or maternal origin. Molecular markers were used to permit evaluation at an early stage, before whole plant development. Somatic embryos developed from potentially apomictic `Sunland' and `Cisco' ovule tissue isolated from bagged putatively unpollinated flowers. Phosphoglucomutase (PGM) isozyme analysis showed that all of these embryos, except one from each cultivar, carry the same zymotype as the maternal tissue. However, restriction fragment length polymorphism (RPLP) analysis combined with isozyme evaluation demonstrated that the tested embryos originated from zygotic rather than maternal tissues. This study demonstrates the application of molecular marker analyses, particularly RFLPs, evaluation of somatic embryo origin.

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Luís Goulão, Luisa Monte-Corvo, and Cristina M. Oliveira

Variability of commercial plum (Prunus L. sp.) cultivars is unknown since breeding often involves intercrossing hybrids with several species but has been based on a low number of parents. Molecular markers like amplified fragment length polymorphisms (AFLP) and inter-simple sequence repeats (ISSR), which sample multiple loci simultaneously, have become increasingly popular, and were used to characterize 24 diploid and four hexaploid cultivars of plum. Seven AFLP and six ISSR primers were used, and resulted in amplification of 379 and 270 products, respectively. Unweighted pair-group method with arithmetic averages (UPGMA) dendrograms, based on similarity coefficients, reflected a clear separation between diploid and hexaploid plums. Among diploid plums, two pairs of cultivars were relatively distinct from the rest, namely `Golden Japan' and `Methley' and `Ozark Premier' and `Songold'. Furthermore, several cultivars were grouped together both with AFLP and ISSR analysis: 1) `Ambra', `Red Beaut', and `Black Beaut', 2) `Black Diamond' and `Royal Diamond', 3) `June Rose', `Santa Rosa', and `Royal Red', and iv) `Freedom', `Larry Ann', and `Queen Rosa'. Although the phenetic classification obtained by the two methods were similar (r = 0.73, for the diploid group), ISSR had a higher reproducibility and percentage of polymorphisms (87.4% vs. 62.8%) than AFLP. Methodological aspects of both markers systems are discussed. Results obtained suggest that the AFLP and ISSR approaches are valuable tools for identification of specific genotypes and analysis of phenetic relationships in plum.

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Roberto F. Vieira, Peter Goldsbrough, and James E. Simon

Molecular markers were used to assess genetic diversity in basil (Ocimum L. spp., Lamiaceae). Using randomly amplified polymorphic DNA (RAPD) analysis, 11 primers generated 98 polymorphic bands, ranging from 300 to 2,000 base pairs, that discriminated among 37 accessions across nine Ocimum spp. Means of genetic similarities within Ocimum spp. showed that the domesticated species, O. minimum L. (0.887), O. basilicum L. (0.769), and O. ×citriodorum Vis. (0.711) had highest similarity indices within species, while the nondomesticated, O. americanum L. (0.580), O. gratissimum L. (0.408), and O. kilimandscharicum Guerke (0.559) showed the lowest similarity. RAPD results indicated that O. minimum should not be considered a distinct species but rather a variety of O. basilicum. Consistent clusters among all but one of the O. ×citriodorum spp., all containing citral as the major constituent, were identified using bootstrap analysis. RAPD analysis was useful in discriminating among Ocimum spp., although within species resolution will require a higher number of polymorphic bands.