Morphological and molecular methods to identify butternut (Juglans cinerea) and butternut hybrids: relevance to butternut conservation

Abstract

Butternut (Juglans cinerea L.) is a native, cold-tolerant, hard-mast species formerly valued for its nuts and wood, which is now endangered. The most immediate threat to butternut restoration is the spread of butternut canker disease, caused by the exotic fungus Sirococcus clavigignenti-juglandacearum Nair, Kostichka & Kuntz. Other threats include the hybridization of butternut with the exotic Japanese walnut (Juglans ailantifolia Carr.) and poor regeneration. The hybrids, known as buartnuts, are vegetatively vigorous, highly fecund, more resistant than butternut to butternut canker disease and difficult to identify. We review the vegetative and reproductive morphological traits that distinguish butternut from hybrids and identify those that can be used by field biologists to separate the taxa. No single trait was sufficient to separate butternut from hybrids, but pith color, lenticel size, shape and abundance, and the presence or absence of a notch in the upper margin of leaf scars, can be used in combination with other traits to identify butternuts and exclude most hybrids. In at least one butternut population, reduced symptoms of butternut canker disease were significantly associated with a dark barked phenotype. We also describe two randomly amplified polymorphic DNA (RAPD) markers that differentiate butternuts from hybrids based on DNA polymorphism. Together, these results should assist in the identification and testing of non-hybrid butternut for breeding and reintroduction of the species to its former habitats.

Walnut: past and future of genetic improvement

“…(pro sp. ), a hybrid between J. cinerea (butternut) and J. regia (Ross-Davis et al 2008). These hybrids are vigorous and more resistant to butternut canker disease, and RAPDs differentiating pure butternut from hybrids were found.…”

DNA markers identify hybrids between butternut (Juglans cinerea L.) and Japanese walnut (Juglans ailantifolia Carr.)

“…A core set of genotypes was evaluated with a speciesspecific chloroplast CAPS marker (CPS05; McCleary et al 2009) to ensure the samples' taxonomic assignment based previously on morphology (Ross-Davis et al 2008b). Trees containing a J. ailantifolia chloroplast but producing a butternut-shaped seed were deemed hybrids; sources that originated from forest habitats, contained a J. cinerea chloroplast, and showed J. cinerea morphology were deemed butternuts.…”

Use of RAPD analysis to assess the threat of interspecific hybridization to the critically endangered Polemonium kiushianum in Japan

“…Among the different types of molecular markers available, random amplified polymorphic DNA (RAPD) analysis requires no prior knowledge of the genome that is being analyzed, and can be employed across species using universal primers (Agarwal et al 2008). Due to their convenience, RAPD markers have been utilized to assess the extent of hybridization, analyze genetic diversity and provide phylogenetic information in many plant species, e.g., butternut (Ross-Davis et al 2008), Flemingia macrophylla (Heider et al 2007), ash gourd (Verma et al 2007) and walnut (Orel et al 2003). The amplification reaction based on the short primers in RAPD analysis is characterized by low stringency, normally achieved with low annealing temperatures (Welsh and McClelland 1990).…”