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Juglans nigra and Juglans regia are economically important species in Europe, Asia and North America. Natural hybrids between the two species, known as Juglans x intermedia (Carr), are valued for timber production. We tested ten nuclear microsatellite markers to (1) identify new J. x intermedia hybrids and characterize their parentage species J. regia and J. nigra (2) detect J. nigra genotypes with a spontaneous crossing ability with J. regia in a mixed Italian population. This study was also designed to confirm the transferability of ten black walnut SSR loci to Persian walnut All ten microsatellites amplified in both species, producing fragments of variable size; eight (7.14%) were common, 68 (60.7%) amplified in J. nigra and 36 (32.1%) in J. regia only (private alleles). Indices of genetic diversity revealed high level of variability. The Principal Coordinate Analysis on the basis of total 112 alleles divided the total sample set into three main groups: J. nigra, J. regia and J. x intermedia hybrids. Performing the microsatellite fingerprinting, a triploid hybrid plant with two genome parts of J. nigra and one part of J. regia was identified. The cytological analysis proved this triploid state showing 48 somatic chromosomes. The mother testing analysis of the 7 diploid hybrids by exclusion method indicated one putative hybridogenic mother plants. The sequence analysis of amplified fragments confirmed the cross-species amplification of SSR. Inter-specific differences between alleles were due not only to simple changes in the number of repeats but also to mutations in the flanking regions.

Characterization of Juglans nigra (L.), Juglans regia (L.) and Juglans x intermedia (Carr.) by SSR markers: a case study in Italy