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Informative DNA fingerprints from 50 walnut cultivars (Juglans regia) in China were generated using amplified fragment length polymorphism (AFLP) markers to reveal their genetic diversity and relationships. Nine primer combinations were selected from 64 EcoR I/Mse I primer combinations to amplify the accessions. An average of 132 polymorphic loci per primer set was detected from the nine primer combinations. The discrimination power of each polymorphic marker (estimated by the polymorphism information content) ranged from 0.00 to 0.37 with an average of 0.19. A moderate level of genetic diversity was observed among the 50 cultivars. Their expected heterozygosity varied from 0.38 to 0.50 (average, 0.44), and Dice's similarity coefficient ranged from 0.53 to 0.86 (average, 0.70). The cluster analysis conducted using the unweighted pair group method of arithmetic averages method showed that all of the cultivars fell into five groups at Dice's similarity coefficient of 0.68. According to the comprehensive analyses based on the specific loci, similarity coefficient, and clustering results, six cultivars (Liaoning 1, Zixin, Shanhe 4, Zha 343, Tulare, and Chandler) were considered important germplasms of walnut cultivars.
Monoclonal anti-indole acetic acid antibodies were used to monitor the temporal and spatial pattern of auxin during staminate flower differentiation in walnut (Juglans regia) cultivars Liaoning 1 and Liaoning 3. The relationship between morphological characteristics and histological structure was established. Seven stages of differentiation were recognized based on the visibility and color of the squama, bract, perianth, and rachis as follows: Stage 1, several bract primordia were present in the squama with catkins protruding from the squama as the only externally visible portion of the floret (Stage 1); the bract became externally visible, and the floret, perianth, and stamen primordia formed basipetally (Stage 2); the length of catkins were elongated, only bracts visible and getting brown (Stage 3); the bracts were brown and wrapped tightly, cellular specialization occurred to form a central core containing reproductive cells and tapetal cells that differentiated (Stage 4); the perianth became visible externally, reproductive cells and tapetal cells separated from the exterior layers of the anther wall (Stage 5); the anther walls were reduced to two cell layers (epidermis and endothecium) as the anthers became visible and matured (Stage 6); and the anther turned black, dehisced, and released its pollen grains (Stage 7). The histological differentiation of the flowers was related to auxin. The auxin signal was strongest in the shoot apical meristem (SAM) during bract primordia differentiation; thus, the SAM may be a site of auxin production. When the floral organs began centralized differentiation, auxin was distributed mainly in the differentiating tissues. Our findings indicate that a high level of auxin may strongly affect morphogenesis. Additionally, the tapetal and reproductive cells that arise during cellular specialization may be important for auxin production. The distribution of auxin was centralized in germ pores at the pollen grain surface, indicating that a high level of auxin induces pollen germination.
We used anti-indole-3-acetic acid (IAA) monoclonal antibodies to monitor the temporal and spatial pattern of IAA during pistillate flower bud differentiation in the walnut (Juglans regia) cultivar Liaoning 1. Based on morphological changes, the process of pistillate flower bud differentiation was divided into five stages. The flower induction stage, which includes the early phase, midphase, and late phase, persisted from 25 Apr. to the end of May. The pedicel differentiation stage began on 5 June. The bract primordium stage began on 25 June and persisted through mid-March of the next year. Both the perianth and pistil differentiation stages persisted for nearly 2 weeks. During the floral induction period, little IAA was present in the shoot apical meristem (SAM); hence, the SAM may not always be a site of IAA production. IAA was obviously concentrated in cells of the first several layers of the SAM during pedicel primordium formation. High levels of IAA were also noted in the phyllome, young leaf tips, and vascular bundle of leaves and gemmae. This direct evidence indicates that no close relationship exists between IAA and physiological differentiation; instead, IAA may strongly affect morphogenesis. These findings comprise a first step toward elucidating the walnut flowering mechanism.
Cotyledon explants of walnut (Juglans regia) have been shown to generate adventitious roots on growth regulator-free medium. The spatial distribution of endogenous indole-3-acetic acid (IAA) and its dynamic changes during adventitious root formation were investigated using an in situ immunohistochemical approach. Before root induction, IAA signal was distributed throughout the freshly excised cotyledon explants. During provascular bundle differentiation, the IAA signal was mainly located in the provascular bundles. At the stage of annular meristematic zones formation, the IAA signal was mainly distributed in the meristematic zones and decreased in the vascular bundles and cotyledonous parenchyma. As primordia formed, the IAA signal became localized in the root primordia and gradually disappeared in the meristematic zones. In emerging roots, the IAA signal was mainly localized in the root cap and root meristem. These results suggest that accumulation of IAA in the provascular bundles may induce vascular differentiation and the increase in IAA through meristematic zones may be responsible for the adventitious root formation from walnut cotyledons. The direct evidence presented here indicates that IAA accumulated in the meristematic zones is not the sole signal needed to induce adventitious root.
Molecular markers were used to study the genetic diversity, structure, and relationship of Juglans L. with nine populations (five from Juglans regia L. and four from Juglans sigillata Dode) in central and southwestern China. A moderate level of genetic diversity was observed at the population level with the number of effect alleles per locus (A E) ranging from 1.75 to 3.35 (average 2.39) and the proportion of polymorphic loci (P) equaling 100.0%. The expected heterozygosity (H E) within populations ranged from 0.389 to 0.687, and the average was 0.525. The proportion of genetic variation presented among populations accounted for 18.6% of the total genetic diversity. The overall gene flow (N m) among populations equaled 1.10. The unweighted pair-group method using arithmetic averages (UPGMA) clustering and the Mantel test showed that genetic distances among the nine populations are in a good agreement with their geographic distribution, supporting the viewpoint that J. regia and J. sigillata belong to one species. We suggest that the central area of the southwestern mountain regions of China could be considered as a priority for walnut genetic resource conservation.