| Walnut is an important non-timber forest tree species. A great amount of excellent walnutcultivars were selected till now. However, there is still a far way to go for walnut geneticdiversity studies. In this research, genetic diversity analysis was carried out on the basis ofmorphology markers, AFLP, and SSR. An advanced AFLP system was established and appliedto analyse the relationships of differenent walnut cultivars. AFLP fingerprintings of136walnutcultivars were conducted. Genetic structures and distances between the tested cultivars weredetermined both at the morphological level and the molecular level. The research has animportant significance in the enrichment of the walnut variety database, the blindness decreaseof walnut breeding and the effective protection of known walnut cultivars. Meanwhile it canimprove the authorization efficiency and accuracy of new walnut cultivars and lay a goodfoundation for walnut germplasm conservation and molecular marker-assisted breeding. Themain results are as follows:1. Morphological variation was found to be giant among different walnut cultivars. Allassessed cultivars generated genetic distance averaged6.4246. Some cultivars were hard todistinguish according to their morphology.2. High quality genomic DNA was isolated from fresh phloem of one-year old branchusing an advanced CTAB protocol.17pairs of primer combinations were screened out in theuse of f-AFLP fingerprints conduction. Fingerprints conducted in the study were robust andrepeatable.3. The f-AFLP fingerprintings of136assayed walnut cultivars were constructed.75cultivars had specific bands and the others could be distinguished by their differential bands inAFLP fingerprinting through the dichotomous classification method. All tested walnut cultivarscould be distinguished according to their specific bands. 4. Seventeen EcoR I/Mse I primer combinations selected with clear and good separationbands and high polymorphism were used for the f-AFLP analysis of136walnut cultivars.3008out of3100bands were polymorphic with an average polymorphism of96.97%. The DICEgenetic coefficients among walnut cultivars were ranged from0.5768to0.8587with anaverage of0.5341. The averaged gene frequency was0.8358, Ne averaged1.3091, H averaged0.2867, I averaged0.1956, which impacted a morderate genetic diversity level of walnutCultivars in China. Dendrogram of assayed walnut cultivars based on f-AFLP data wasconstructed by UPGMA method. The relationships and genetic diversity between them werecarried out at the molecular level. The results revealed the complicated genetic background ofthe walnut cultivars in China which could provide the theoretic basis for the parent selection,cultivar identification and property right protection, and the comparison between cultivars.5. Twenty out of136walnut cultivars were selected as core collection using a methodcombining f-AFLP and morphology. T-test showed there was no significant difference betweenthe core collection and initial germplasm on0.01level. The core collection could representinitial germplasm.6. Clonal cultivars selected from a broad geographic range in China including4types,Juglans regia, Juglans sigillata, Juglans sigillata×Juglans regia, and J. hopeiensis, wereapplied in f-AFLP analysis. Nine EcoR I/Mse I primer combinations produced clear-cut AFLPprofiles. The percentages of polymorphic bands (PPBs), observed number of alleles (Na),effective number of alleles (Ne), Nei's gene diversity (H), and Shannon's information index (I)were calculated based on AFLP data. As for Juglans regia, Juglans sigillata, and their hybrids,PPBs averaged93.86%,93.94%, and60.46%; Na averaged1.5370,1.6389, and1.2593; Neaveraged1.2582,1.2543, and1.1646; H scored0.1560,0.1556, and0.0951; I averaged0.2402,0.2459, and0.1414, respectively. The overall levels of Na, Ne, H, and I were1.7407,1.2701,0.1667, and0.2640. The total gene diversity (Ht=0.1596) can be distributed into intra-(Hs=0.1356) and inter-(Dst=0.0240) species gene diversity. Proportion of the distributed geneticdiversity among populations (Gst) was0.1505represented that only15%of the total gene diversity was attributed to differences between species. The FCA and the UPGMA analysisbased on cultivar polymorphism AFLP data revealed a really close genetic distance betweenJuglans regia and Juglans sigillata.7. SSR markers were applied to analysis of genetic diversity of walnut cultivars. Totally,52alleles were detected generated a PPB of100%. The total value of observed heterozygosity(Ho) and expected heterozygosity (He) within species were0.3977and0.6152. A moderatelevel of genetic diversity was observed. Genetic stucture of assayed cultivars was analysedusing a popgene software.8. Paradox is a famous rootstock in the walnut industry for a number of important features,including fast growth and resistance to some diseases. Paradox commonly refers to blackwalnut-Persian walnut hybrid. Nuclear rDNA IGS region, typically bi-parentally inherited,with rapid evolution and broad existence in all eukaryotic genomes, was found to be ofimportance in revealing genetic background of the walnut hybrids. Both parental geneticcomponents (around87.50%to88.89%) and novel genetic components (around11.11%to12.50%) were detected in the nrDNA IGS8-ETS1region of the Paradox genome. Theinheritance was commonly one-parent-dominated in each hybridization event. Our resultsindicated that genetic formation of Paradox hybrids involved in J. regia (sect. Juglans) and thefollowing6black walnut species (sect. Rhysocaryon), i.e., J. hindsii, J. californica, J. major, J.nigra, J. microcarpa and J. hirsuta. The nrDNA IGS8-ETS1region is helpful in understandingthe genetic basis of hybrids.
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