Microsatellites are an important class of DNA marker because of their abundance and length hypervariability. As part of a project mapping the Pinus radiata genome, we have characterized some of the microsatellites in this species. Southern blots were screened with oligonucleotide probes [(CA)10, (GA)10, (GAA)9, (CAA)8, (CAC)5, (GACA)4] to assess their abundance. CA and GA were the most abundant microsatellites, while GAA was least abundant. A genomic library in lambda ZAP, covering 9 x 10(4) kb, was screened with a combined poly(CA) + poly(GA) probe and yielded 120 positives, approximately one CA or GA microsatellite every 750 kb of the P. radiata genome. It was found that 25% of the positives were embedded within highly repetitive DNA. Four of the five subclones sequenced contained compound microsatellites, with TA predominating as the additional repeat. Segregation analysis of PCR products for two microsatellites, PR4.6 and PR9.3, in 96 progeny of a controlled outcross verified simple Mendelian inheritance. Both loci are highly polymorphic with Polymorphism Information Content values of 0.63 and 0.70 for PR4.6 and PR9.3, respectively. These results indicate that microsatellites are abundant in a conifer genome and can be valuable markers for pine mapping, fingerprinting, and population genetic studies.

Agrobacterium tumefaciens is established as a vector for gene transfer in many dicotyledonous plants but is not accepted as a vector in monocotyledonous plants, especially in the important Gramineae. The use of Agrobacterium to transfer genes into monocot species could simplify the transformation and improvement of important crop plants. In this report we describe the use of Agrobacterium to transfer a gene into corn, the regeneration of plants, and detection of the transferred genes in the F1 progeny.Shoot apices of Zea mays L. variety Funk's G90 were cocultivated with A. tumefaciens EHAl, which harbored the plasmid pGUS3 containing genes for kanamycin resistance (NPT II) and ,B-glucuronidase (GUS). Plants developed from these explants within 4 to 6 weeks. Fluorometric GUS assays of leaves and immature seeds from the plants exhibited low GUS activity. Both NOS and GUS gene fragments were amplified by polymerase chain reaction in the DNA isolated from the F1 generations of one of the original transformed plants. Southern analysis showed both GUS and NPT probes hybridized to DNA in several of the F1 progeny, demonstrating the incorporation of GUS and NPT II genes into high molecular weight DNA. These data establish successful gene transfer and sexual inheritance of the genes.Until recently, the monocotyledons and particularly the graminaceous crop species have been considered to be outside the Agrobacterium host range (1, 5). In the past, a general definition of host species range has been based on tumor or gall formation in inoculated plants. Gene transfer methods developed for economically important species considered to be outside of the Agrobacterium host range have previously been restricted to the direct transfer of DNA into protoplasts and to the few cultivars which can be regenerated from protoplasts. With the development of the particle discharge or acceleration methods of direct DNA transfer, intact cells of embryogenic callus and cell suspensions can be used. Recently, this approach resulted in the successful transformation and regeneration of corn (7,10). This approach will be applicable to maize genotypes which form embryogenic cultures.The host-range ofAgrobacterium has been under continual revision since the original review by DeCleene (5). Upon

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 1∶1 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.

GeneticsRandom amplified polymorphic DNA markers tightly linked to a gene for resistance to white pine blister rust in sugar pine ( ABSTRACTWe have genetically mapped a gene for resistance to white pine blister rust (Cronartium ribicola Fisch.) in sugar pine (Pinus lambertiana Dougl.) by using an approach which relies on three factors: (i) the ability to assay for genetic markers in the haploid stage of the host's life cycle, using megagametophyte seed tissue; (ii) a simple and clearly defined pathosystem; and (iii) the use of random amplified polymorphic DNA (RAPD) markers that can be quickly and efficiently evaluated. Resistance to white pine blister rust in sugar pine is known to be controlled by a single dominant gene (R). Maternal segregation ofR and dominant RAPD markers were scored simultaneously following collection of megagametophytes for DNA assays and seedling inoculation with C. ribicola. Bulked samples of haploid megagametophyte DNA from resistant and susceptible offspring of segregating full-sib and half-sib families were used to evaluate 800 random decanucleotide primers. Ten loci linked with the gene for resistance to white pine blister rust were identified and segregation data were obtained from five families. Six of the linked markers were within 5 centimorgans of the gene, and one marker was 0.9 centimorgan from R. These and other markers derived by this approach may provide starting points for map-based cloning of this important gene.

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