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Mei Guo, David A. Lightfoot, Machteld C. Mok, and David W. S. Mok

142 ORAL SESSION (Abstr. 666-672) CROSS-COMMODITY BIOTECHNOLOGY II/RFLPs

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Mei Guo, David A. Lightfoot, Machteld C. Mok, and David W. S. Mok

142 ORAL SESSION (Abstr. 666-672) CROSS-COMMODITY BIOTECHNOLOGY II/RFLPs

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Wayne Kennard, Arian Dijkhuizen, Michael Havey, and Jack Staub

142 ORAL SESSION (Abstr. 666-672) CROSS-COMMODITY BIOTECHNOLOGY II/RFLPs

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R. Fjellstrom and D.E. Parfitt

RFLP probes were developed to determine the degree of genetic diversity both within and between 12 walnut species (Juglans spp.), including the widely cultivated English walnut (J. regia). One to three kilobase DNA fragments from Pst I digested J. regia nuclear DNA were cloned using the vector pUC18. Inserts corresponding to low copy number walnut genomic sequences were used to assess the genetic variability among walnut species. Extensive polymorphism was found between species and limited polymorphism within species. The inheritances of the RFLP loci are being analyzed to provide a genetic basis for the polymorphisms detected and to establish a RFLP based linkage map in walnuts

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David C. Jarrell and Mikeal L. Roose

We report a preliminary genetic map of citrus based on segregation of 8 isozyme and at least 33 RFLP loci. The segregating population consisted of 60 plants from a cross of two citrus rootstock, `Sacaton' citrumelo × `Troyer' citrange. This cross represents an intergeneric F2 since `Sacaton' is Citrus paradisi (grapefruit) × Poncirus trifoliata (trifoliate orange) and `Troyer' is C. sinensis (sweet orange) × P. trifoliata. RFLPs were identified using anonymous probes from both cDNA and genomic DNA libraries of citrus. About 20% of the loci deviated significantly from Mendelian segregation. Two-point linkage analysis identified 8 linkage groups in which pairs of loci were within 30 centimorgans. This suggests that we have markers on most of the 9 chromosomes of Citrus. A map based on multipoint linkage estimates will be reported. Evidence for structural rearrangements between Citrus and Poncirus and extension of the map to additional marker and disease resistance loci will be discussed.

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L. Eldredge, R. Ballard, W.V. Baird, A. Abbott, P. Morgens, A. Callahan, R. Scorza, and R. Monet

Peach [Prunus persica (L.) Batsch.] is considered the best genetically characterized species of the genus Prunus. We therefore used it as a model in our study of the genome organization in Prunus by means of restriction fragment length polymorphisms (RPLPs). Initial results indicated that 60% of cloned DNA sequences examined occur at low copy number within the peach genome. After selecting and examining these sequences, polymorphisms sufficient for RPLP mapping were found. We determined that ≫33% of our cDNA clones and 20% of our genomic clones detected RPLPs among peach cultivars. Analysis of RPLP segregation in two families, both of which segregate for known morphological characters, revealed segregation in 12 RFLP markers for one family and 16 for the other. Although we have not detected linkage between RFLP and morphological markers, preliminary analyses indicate possible linkage between two RPLP markers.

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Robert G. Fjellstrom and Dan E. Parfitt

32 cloned probes from a walnut (Juglans sp.) PstI random genomic library were used to develop a linkage map for walnut. Low copy number walnut random genomic DNA probes were constructed and hybridized to restriction endonuclease digested DNA from parent walnut trees from a backcross of (J. hindsii × J. regia) with J. regia to identify parental polymorphism. 63 backcross progeny were analyzed to determine the inheritance and linkage of 48 RFLP loci. 66% of the probes detected duplicated, but unlinked loci. 42 of the RFLP loci could be placed on 12 linkage groups. The other 6 loci could not be placed on common linkage groups. (Theoretical maximum number of linkage groups is 16.) A Poisson probability method for estimating genome size was utilized to calculate the approximate walnut genome length as 1660 cm and to estimate that 138 markers would be needed to cover 95% the walnut genome within 20 cM of each marker.

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Robert G. Fjellstrom, Dan E. Parfitt, and Gale H. McGranahan

RFLP markers were used to study genetic diversity among California walnut (Juglans regia L.) cultivars and germplasm collected worldwide. 16 of 21 RFLP markers were polymorphic in the 48 walnut accessions tested. Seven RFLP markers permitted unique identification of all walnut cultivars. All genotypes were heterozygous at approximately 20% of the loci for both California and worldwide germplasm. California walnut germplasm contained 65% of the worldwide allelic diversity. Cluster analysis of genetic distance between accessions and principal component analysis of allelic genotypes showed two major groups of walnut domestication. California germplasm was associated with germplasm from France, Central Europe, and Iran, and had less genotypic similarity with germplasm from Nepal, China, Korea, and Japan.

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Pan-chi Liou, Fred G. Gmitter Jr., and Gloria A. Moore

Citrus genetic studies and cultivar improvement have been difficult with conventional techniques. Alternative approaches are needed to enhance efficiency of such studies. Our objectives were to characterize the Citrus genome and to initiate development of a linkage map using RFLP and isozyme analysis. Methods of Citrus DNA extraction were developed to allow the isolation of chromosomal DNA of acceptable quality for recombinant' DNA manipulations. A PstI Citrus genomic library was constructed to create DNA clones for the RFLP survey. A rapid, reliable procedure was developed to facilitate screening of the library for useful clones. The methods used and strategy followed minimized contamination with organelle DNA, increased the frequency of single copy clones, and allowed rapid screening of the newly–constructed library. Linkage relationships of 49. markers, including 36 RFLP and 6 isozyme loci, were analyzed and a map comprised of 8 linkage groups was constructed. Insertions or deletions were responsible for at least 30% of the RFLPs identified. A hypothesis of transposon activity in Citrus was proposed based on our observations.

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R. Fjellstrom and D. E. Parfitt

RFLP analysis was employed to study the inheritance of and genetic diversity identified by cloned walnut genomic probes. An interspecific backcross population of (J. hindsii × J. regia) × J. regia was used to determine the inheritance of thirty low copy number RFLP cloned probes. Of these probes, approximately 20% correspond to single copy loci, 40% correspond to single major loci with multiple minor loci, and 40% correspond to two major loci. Twenty of these probes were used to analyze variability within and between 13 walnut species (Juglans spp.). Substantial genetic variation was identified within many wild walnut species, while limited variation was identified within butternut (J. cinerea) and the widely cultivated English walnut (J. regia). Extensive polymorphism was found between walnut species, allowing a phylogenetic relationship of walnuts based upon RFLP markers to be developed. Identification of clonally propagated walnut cultivars by RFLP typing was readily performed in black walnut (J. nigra) accessions, was more difficult in English walnut accessions, and rarely possible in butternut accessions.