BREEDING, CULTIVARS, ROOTSTOCKS, & GERMPLASM RESOURCES HORTSCIENCE 38(6):1178–1183. 2003. genetic similarity of the component elements, can be of practical advantage. Our long term goal is to develop superior rootstocks for Jug- Characterization of 11 Juglandaceae lans trees for timber production. The fi rst step towards that goal was to infer phy lo ge net ic Gen o types Based on Morphology, relatedness between our accessions based on morphology, cpDNA, and RAPD analysis cpDNA, and RAPD presented in this study. 1 G. Orel Materials and Methods Centre for Horticulture and Plant Sciences, University of Western Sydney, Hawkesbury Campus, Locked Bag 1797, South Penrith DC, NSW 1797, Accessions used (Table 1).Juglans nigra L. and J. olanchana Standl. et L.O. Williams are Australia from temperate and subtropical North Amer i ca, National Herbarium of New South Wales, Royal Botanic Gardens Sydney, Mrs respectively, and J. neotropica Diels. and J. australis Griseb. from tropical and temperate Macquarie·s Road, Sydney NSW 2000, Aus tra lia South America. Juglans nigra is a well-identi- A.D. Marchant fi ed cultivated species, while the other three are new germplasm accessions. Juglans regia National Herbarium of New South Wales, Royal Botanic Gardens Sydney, Mrs is of Eurasian origin; J. sigillata Dode and an Macquarie·s Road, Sydney NSW 2000, Aus tra lia undescribed species (“J. sp.”) from China, and J. ailantifolia Carr. from Japan. Engelhardia J.A. McLeod and G.D. Richards spicata Leschenault ex Blume is from Royal Centre for Horticulture and Plant Sciences, University of Western Sydney, Botanic Gardens, Sydney, of Viet nam ese prov- Hawkesbury Campus, Locked Bag 1797, South Penrith DC, NSW 1797, enance. Another (unidentifi ed) Engelhardia Australia species was collected in Yunnan province, China. The North American Carya illinoensis Additional index words. chloroplast, graft, interspecifi c, Juglandaceae, morphology (Wangh.) K. Koch. specimen is from the living collection of the Univ. of Western Sydney. Abstract. The interspecifi c and in ter ge ner ic relationships of eight species of Juglans All sequences were generated during this (wal nuts) and three other members of Juglandaceae were investigated. The following study except for Juglans cathayensis Dode. species were included: the American J. australis Griseb., J. neotropica Diels., J. olanchana The trnL intron sequence of J. cathayensis, Standl. et L.O. Wil l iams, J. nigra L., and Carya illinoensis (Wang.) K. Koch.; two Juglans a Chinese species, was imported from Gen- from South China, namely, J. sigillata Dode and an un i den ti fi ed J. sp; an Engelhardia Bank (Accession No. AF 200936; Wu et al., also from China and the Asian J. ailantifolia Carr., Pterocarya stenoptera var. tonkinensis 1999). Franchet and the Eurasian J. regia L. Cla dis tic analysis of 27 multistate mor pho log i cal DNA extraction and purifi cation. Ge nom ic characters showed that the juvenile J. ailantifolia possessed similar physical traits to DNA was extracted from fresh leaves, using that of the juvenile American Juglans species. The chloroplast DNA in the trnL–trnF a method similar to that of Dellaporta et al. region indicated a close relationship between Juglans species. Pterocarya put the root of (1983, as described by Wilkie, 1997), fol lowed the cpDNA network among the American species. RAPD analysis was performed using by purifi cation using diatomaceous earth bind- eight primers. A total of 138 fragments were generated but only 78 clearly defi ned bands ing, adapted from the technique described by were used in the analysis. All the DNA data grouped the tropical/subtropical American Gilmore et al. (1993). Juglans with J. nigra, and the two new Asian species with J. ailantifolia and J. re gia. The Polymerase chain reaction (PCR.). A region American species were closely re lat ed, more so than their Asian coun ter parts. The close- of chloroplast DNA comprising the tRNA leu- ness of the investigated spe cies predicts interspecifi c graft com pat ibil i ty not only within cine (UAA) gene (trnL), the intron it contains, the Asian and American groups, but also between them. the tRNA phenylalanine (GAA) gene (trnF), and the intergenic spacer between trnL (5´ Juglandaceae contains six genera and 60 ter of origin (Manchester, 1987). Manchester exon) and trnF, was amplifi ed by PCR (Mullis species (Bhattacharyya and Johri, 1998; Stace, argued that the Juglandaceae orig i nat ed in the and Floona, 1987), using the primers A50272 1996). It has been divided into two subfamilies, Northern Hemisphere, with a major diversifi ca- and B49317 of Taberlet et al. (1991). Reac- Platycaryoideae and Juglandoideae, and these tion occurring during the Paleocene. This view tions were performed in a HYBAID OMN-E subfamilies have each been divided into two was reinforced with the discovery of the extinct thermocycler, using the following program: 5 tribes (Manches ter, 1987). Of the four genera Juglandaceous spe cies, Polyptera manningii, min at 94 °C; 30 cycles of 30 s at 94 °C; 30 s examined in this study, Engelhardia belongs to in Paleocene de pos its in Wyoming and Mon- at 60 °C and 1 min at 72 °C. the tribe Engelhardieae in Platycaryeae, Carya tana (Manchester and Dilcher, 1997). The reaction mixture contained 2.5 µL to the tribe Hicoreae in Juglandoideae, and Grafts between the varieties of same spe- 10× PCR buffer (Promega #M190G), 1.5 µL Juglans and Pterocarya to the tribe Juglandieae cies, and between different species within the 25 mm MgCl2, 2 L ¶dNTPs· (2.5 mm each of (Manches ter, 1987; Yong-Ling et al., 1992). same genus, account for the majority of all dATP, dCTP, dGTP, and dTTP), 5 µL of each Juglandaceae are distributed in two geo- grafts performed in horticultural industries of the two primers at a concentration of 20 graphically distinct areas—in the Amer i cas, (Dirr and Heuser, 1987; Ross, 1997; Thain µm, 8 µL H2O, and 0.1 µL Taq Polymerase (5 and in Eurasia, from Central Europe to Japan and Hickman, 1995). Graft compatibility units/µL, Promega). PCR products were pu- (Bhattacharyya and Johri, 1998; Krussmann, be tween species in different genera, or even ri fi ed using the CONCERT PCR Purifi cation 1985; Leroy, 1953; Manning, 1960). Eastern different families, is also known. In com mer cial Kit (GibcoBRL Co.). DNA sequences were Asia is considered to be the current center of horticulture, a particular concern is “de layed ascertained by the Univ. of Sydney and Prince diversity for the Juglandaceae, but not the cen- graft incompatibility.” In some in stanc es, grafts Alfred Molecular Analysis Centre, Sydney, will take initially, but rejection and consequent Australia, using the ABI ¶Prism· fl u o res cent death (or at least debility) of the scion, may dye-terminator system (Applied Biosystems, Received for publication 23 July 2002. Accepted occur several years after the graft was ini- Foster City, Calif.). for publication 4 Feb. 2003. tially performed. So, having an in di ca tion of Random Amplifi cation of Polymorphic 1To whom reprint requests should be ad dressed. the likely long-term success of various grafts, DNA (RAPD). The protocol of Welsh and E-mail: [email protected] which can be provided by an indication of the McClelland (1990) was followed. DNA 1178 HORTSCIENCE, VOL. 38(6), OCTOBER 2003 1178-1183 1178 10/24/03 10:35:29 AM Table 1. Provenance details of Juglandaceae specimens used in this study. Taxon Voucher no.z GenBank no. Wild source Cultivation sitey Juglans ailantifolia Carr. NSW 476462 AY231169 unknown Mt Irvine Juglans australis Griseb. NSW 476464 AY231171 Tucuman, Argentina Nambucca Headsx Juglans neotropica Diels. NSW 476473 AY231168 Loja, Ecuador Nambucca Heads Juglans nigra L. NSW 476485 AY231170 unknown Cowra Juglans olanchana Standl. & L.O. Williams NSW 476481 Turialba, Costa Rica Nambucca Heads Juglans regia NSW 476488 AY231167 unknown Cowra Juglans sigillata Dode NSW 476484 AY231170 Yunnan, China Nambucca Heads Juglans sp. NSW 476489 AY231172 Quang Xi, China Nambucca Heads Carya illinoensis (Wangh.) K.Koch. NSW 476493 unknown Nambucca Heads Pterocarya stenoptera var. tonkinensis Franchet NSW 476491w AY231174 Sapa, Vietnam RBGv, Sydney Engelhardia sp. NSW 476494 Yunan, China Nambucca Heads zNational Herbarium of New South Wales, Royal Botanic Gardens, Sydney, Australia. yAll of these sites are in New South Wales, Australia. xAnnex of Univ. of Western Sydney, Hawkesbury Campus, Sydney, Australia. wCollected by Simon Goodwin, RBG, Sydney, Australia; all others collected by Orel and/or Richards. vRoyal Botanic Gardens, Sydney, Australia. frag ments of Juglans and outgroup were proportion of fragments, using the formula: state changes of some rel e vant morphological amplifi ed using the following primers: OPA5 characters (Fig. 3). The characters designated 1 – 2N /(N + N ) (AGGGGTCTTG), OPA7 (GAAACGGGTG), xy x y 13, 21, and 27 are plesiomorphic in J. ailan- OPA10 (GTGATCGCAG), OPA18 (AG- where Nxy is the number of bands shared by tifolia. Character 22 reverts in J. ailantifolia, GTGACCGT), OPA19 (CAAACGTCGG), specimens x and y, and Nx is the number of uniquely among Juglans species. A parallel and OPA 20 (GTTGCGATCC) (QIAGEN bands from specimen x (based on Upholt, 1977; reversion is ap par ent in J. ailantifolia and J. OPERON P/L). as cited by Avise, 1994). neotropica (char ac ter 20). A parallel change in The reaction mixture for RAPDs con sist ed leaf surface tex ture is shown in J. ailantifolia × of: 2 µL 10 PCR buffer, 2 µL MgCl2 25 mm, 2 Results and J. neotropica (character number 14) and µLdNTP, 4 µLprimer (20 mm), 10 µLH2O, 0.1 another parallel change of indumentum texture µL of Taq polymerase (5 units/µL, Promega), Morphology. Cladistic analysis of the (19) in J.