Identification of Pinus elliottii var. elliottii X P-caribaea var. hondurensis hybrids using the chloroplast trnL-F intergenic spacer

Breeding programs based on hybrids of tree species often have a need to unambiguously distinguish intraspecific and self progeny from hybrid progeny. The interspecific hybrid of P. elliottii var. elliottii (PEE) and P caribaea var. hondurensis (PCH) is difficult to reliably distinguish from pure PEE based on morphology, especially at a young age. We examined the trnL-F intergenic spacer region of the paternally inherited chloroplast genome for a polymorphism that may distinguish these two taxa. Sequencing this region indicated there were two haplotypes, one which was specific to PEE, designated (+), the other designated (-), was shared by both taxa. This result was consistent with other studies which suggest past introgression of PCH into PEE. A PCR assay was developed to detect the PEE specific haplotype. This haplotype was found at a frequency of 0.6 in a sample of 22 PEE from a breeding population but was absent from a. sample of 30 PCH parents. As expected, hybrids from crosses of PCH pollen donors and maternal (+) haplotype PEE had (-) haplotypes. In situations where the mother can be genotyped, and the pollen pool consists of maternal (self) or PCH pollen, this assay would unambiguously determine the hybrid status of offspring from 60% of the PEE parents tested. Where paternal candidates include other PEE, the assay would conclusively identify non-hybrid progeny at a lower percentage determined by the frequency of the (+) haplotype in the pollen pool. The chloroplast trnL-F spacer assay would be useful for verifying hybrids from future seed orchards based on mother trees with (+) haplotype or for (+) haplotype mother trees in current orchards. The assay will also confirm suspected pure PEE individuals where they have a (+) haplotype but will be inconclusive where they have a (-) haplotype