Polymerase chain reaction identification of a female-specific genetic marker in Arceuthobium americanum (lodgepole pine dwarf mistletoe) and its implications for Arceuthobium sex determination

In North America, the most widespread and speciose mistletoe is Arceuthobium M. Bieb. (dwarf mistletoes, Viscaceae), which is a dioecious parasite of conifers. Little is known about its sex determination system, and sex chromosomes have not been identified. A genetic marker for early gender discrimination in Arceuthobium would be useful in the study of sex ratios and sex determination. Here, random amplified polymorphic DNA analysis via the polymerase chain reaction (PCR) was used to investigate genetic differences between genders in Arceuthobium americanum Nutt. ex Engelm. collected near Kamloops, British Columbia and Bélair, Manitoba. A total of 196 10-mer primers were selected for analysis of DNA from isolated male and female A. americanum somatic tissue. A ∼900 bp female-specific DNA fragment was generated with primer OPB-18 (5′-CCACAGCAGT-3′). The fragment was cloned and sequenced. Using GenBank and the basic local alignment search tool alignment software, it was determined that the first ∼300 bp of this DNA sequence shared a high degree of similarity to transposable elements (76%) and a Y-chromosome (male) fragment (75%) in Silene latifolia Poir. Sequence-characterized amplified region primers were then designed. This study has generated an efficient molecular tool to differentiate male and female A. americanum while also providing evidence indicating that A. americanum may have homomorphic, possibly protoheteromorphic, sex chromosomes.