Genetic recombinational and physical linkage analyses on slash pine.

Abstract

Slash pine is native to the southeastern USA, but is commercially valuable world-wide as a timber-, fiber- and resin-producing species. Breeding objectives emphasize selection for fusiform rust disease resistance. Identification of markers linked to genetic factors conditioning specificity should expand our knowledge of disease development. Towards this end, random amplified polymorphic DNA (RAPD) markers were identified and mapped in a tree hypothesized to be homozygous dominant for resistance at one locus and homozygous recessive at another. Because the DNA prepared for analysis was from haploid maternally-inherited, megagametophyte tissue of seeds, RAPD markers were observed as either present or absent. The analysis revealed 13 linkage groups of three or more loci, ranging in size from 28 to 68 cM, and nine linked pairs. The 22 groups and pairs included 73 RAPD markers and covered a genetic map distance of approximately 782 cM. Genome size estimates, based on linkage data, range from 2,880 to 3,360 cM, and equal 6.0-6.9 x 10(6) bp/cM (physical size > 20,000 Mbp). Using a 30 cM map scale and including unlinked markers, ends of linkage groups, and linked pairs, the RAPD markers account for approximately 2,160 cM or 64-75% of the genome. Mapping 80 additional RAPD markers placed 131 loci total in 20 linkage groups of three or more loci, nearly doubling the coverage in the groups to a genetic map distance of approximately 1,347 cM. Two other slash pine trees also have been RAPD mapped. DNA-DNA in situ hybridization and cytochemical staining are being used to integrate the genetic recombinational maps. A karyotype and ideogram have been prepared for slash pine (2n = 2x = 24); metaphase chromosome preparations show 11 pairs of long metacentric chromosomes and one shorter pair of submetacentric chromosomes. Patterns of fluorescence in situ hybridization to genes for the large and small rRNA subunits and fluorochrome banding patterns using the GC-base-specific chromomycin A3 (CMA) and AT-base-specific 4',6-diamidino-2-phenylindole (DAPI) allowed all twelve pairs of chromosomes to be identified and a standard karyotype established. A family of sequences associated with (TTTAGGG)n related repeats has been identified in slash pine using a labeled synthetic oligonucleotide probe. Fluorescence in situ hybridization shows a weak signal at telomeres and significantly stronger intensity at non-telomeric sites. The most common non-telomeric location was in the pericentric regions of chromosomes; interstitial sites of hybridization were relatively common. Microsatellite DNAs, an abundant retrotransposon-like element, and total genomic in situ hybridization and species and chromosome specific DNAs are being evaluated for analyses of interspecific hybrids and chromosome evolution between related species. Interest in low and single copy sequences is increasing.