E&nding the Karyotype of Slash Pine as a Prelude to Physical Mapping

--Cytological exploration of the pine genome has been ongoing for more than a century. For the first seventy years we knew little more than chromosome number for pines. Constancy in chromosome number throughout the genus coupled with uniformity in size and morpholo,y between chromosomes within species has given cytologists few practical means by which to distinguish individual chromosomes in Pinus (Sax and Sax 1933). Several Karyotypes were published between 1950 and 1970 based in tionstriction patterns in chromosomes in haploid tissues but were not reproducible in metaphase spreads f?om diploid tissues making them of limited utility for routine karyotyping. The task of karyotyping became even more formidable when pines proved recalcitrant to standard chromosome banding procedures like giemsa., DAPI and CMA (Pedrick 1970; Bonan and Papes 1973). The advent of fluorescent in situ hybridization, or FISH, in the last decade has finally made the pine genome visually accessible in the microscope. In the FISH protocol, labeled probes are hybridized to spread metaphase chromosomes which are affixed to glass microscope slides. Sites of probe hybridization on the chromosomes are detected in an epifluorescence microscope after application of a fluorophore conjugated to an antibody specific to the label on the probe. The microscope image is .one of br@t ly lit bands of color against a dark background that is reminiscent of lights on a Christmas tree. In 1995, Do&rick et. al. published the first FISH Karyotype for pine using metaphase spreads from slash pine. These authors also discussed the potential utility of FISH for physically mapping the many genetic linkage groups &at have been identified for slash pine. Implementing physical mapping using this karyotype is problematic because it requires two ribosomal probes and banding by two different fluorochromes for diagnostic karyotyping. This necessitates stripping and reprobing which is labor -htensive and time consuming and also limiting in terms of the number of new probes that can be placed on the existing karyotype. Reported below are recent expansions to this karyotype that could accelerate routine karyotyping and thus serve as a prelude to physical mapping in this species. In the original karyotype, Do&rick et. al. used a heterologous probe from sugar beet to locate 5s rDNA sites. Three chromosomal locations were defined by this probe. In the present study, a homologous probe specific for the 5s rDNA coding region was generated and labeled by PCR using primers PI and P2 from Brown and Carlson (1997) . Using this probe, an additional 15 sites w&e defined , a 5-fold increase in available bands for karyotyping and a 2-3fold increase in the number of chromosomes with ” detectable 5s rDNA sites. :I 7;. .x .aExperiments in which the Telomere Repeat Sequence, or TRS, from Arabidopsis thaZZiana (‘Rich~dS ~~ and Ausubel 1988) was used as a probe, generated bands on all slash pine chromosomes. In addition to hybridization at the telomere, there are an additional 36 interstitial and centromeric bands defined by &is probe with a range of 2-6 bands per chromosome. By comparison, there are a total of 46 bands in the existing karyotype excluding the 6 majo;. constrictions which are seen as negative bands or cleared regions. Of these 46 bands, 16 ar ‘Sohem hd’ute ofForest Genetics, USDA Forest Service, Saucier, MS 39574