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The eastern Asian and eastern North American disjunction in Juglans offers an opportunity to estimate the time since divergence of the Eurasian and American lineages and to compare it with paleobotanical evidence. Five chloroplast DNA noncoding spacer (NCS) sequences: trnT−trnF, psbA−trnH, atpB−rbcL, trnV-16S rRNA, and trnS-trnfM and data from earlier studies (matK, ITS, and nuclear RFLP) were used to reconstruct phylogeny and to estimate the divergence time of major lineages. Seventeen taxa from four sections of Juglans and two outgroup taxa, Pterocarya stenoptera and Carya illinoiensis were included. NCS data was congruent only with matK data. Both maximum parsimony (MP) and maximum likelihood (ML) cladograms were concordant at the sectional level and revealed three well-supported monophyletic clades corresponding to sections Juglans, Cardiocaryon, and Rhysocaryon in both NCS and combined analyses. The single extant American butternut, Juglans cinerea was placed within the poorly resolved, but well-supported Rhysocaryon. Placement of taxa within Rhysocaryon and Cardiocaryon were inconsistent between NCS and combined analyses. Overall, the results suggest that: (1) the NCS sequence divergence observed within and between sections of Juglans is low and the addition of matK data only marginally improved resolution within Rhysocaryon; (2) the early divergence of section Juglans in both MP and ML analyses of NCS and combined data implies its ancient origin in contrast to fossil evidence, which suggests the earliest divergence of sections Rhysocaryon and Cardiocaryon; and (3) the extant taxa may not hold the footprints to unravel the evolutionary history of the genus.

One of the most important rootstocks in the California walnut industry is Paradox, which refers to the offspring of a California black walnut pollinized by a Persian walnut ( Juglans regia ). Paradox was developed by Luther Burbank, who did not distinguish between northern ( J. hindsii ) and southern ( J. californica ) California black walnut. Although it is generally accepted that Paradox designates hybrids between J. hindsii and J. regia , the name is commonly applied to any black walnut–Persian walnut hybrid. Moreover, due to gene flow among black walnut species, the genealogy of Paradox hybrids may also include species such as Arizona ( J. major ) and Eastern black ( J. nigra ) walnut. Since the nuts from which Paradox seedlings are grown are collected from wild trees, their genetic backgrounds are not generally known. In conjunction with a large study aimed at evaluating Paradox hybrids from different industry sources, we have been working to develop molecular markers that can be used to infer the parentage of individual Paradox seedlings. Representatives of the five black walnut species from North America were screened for variability in the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA and in three noncoding regions from the chloroplast genome, the trn T– trn L, trn L– trn F, and trn D– trn T spacers. Unique sequence markers were identified for each species, and total DNA extracts from 27 Paradox source trees were tested for those markers. Chloroplast DNA profiles were used to trace the maternal lineages of the Paradox source trees, while the ITS data provided evidence as to whether or not the source trees were themselves hybrids. Our results indicate that, among industry Paradox sources, there is considerable genetic contribution from species other than J. hindsii .

The utility of intersimple sequence repeat (ISSR) markers for identification of English or Persian walnut (Juglans regia L.) cultivars was explored. Four cultivars were screened with 47 ISSR primers; eight of these primers, which generated reproducible and informative data, were selected for further study. Two individuals from each of 48 cultivars, including many currently important in the California walnut industry as well as accessions from Europe and Asia, were then examined with the eight ISSR primers. Polymerase chain reaction (PCR) products were separated on agarose gels and stained with ethidium bromide. Fifty-four bands were scored as present or absent in each cultivar; of these, 31 (57%) were polymorphic among the 48 cultivars. Combined data from the eight ISSR primers provided a unique fingerprint for each of the cultivars tested. Fifteen of the cultivars could be distinguished from all others with just one primer, 31 with a minimum of two primers, and two required three primers. Pairwise genetic distances between the cultivars were calculated and a dendrogram was generated using the neighbor-joining algorithm. Some of the groupings in the dendrogram corresponded to groups which, based on known pedigrees, are genealogically closely related. Others included accessions from diverse genetic and/or geographic origins. These results can be attributed to a combination of the limitations of the ISSR method for inferring genetic relationships, on the one hand, and the complex history of walnut cultivar development involving extensive exchange and breeding of germplasm from different geographic regions, on the other.