| The genus Juglans (Juglandaceae) consists of about 21 species, many of which are of biologically and economically important because of their high quality timer and nutritious nuts. There are rich germplasm resources for Juglans. Butternut (J. cinerea L.) is a temperate deciduous hardwood native to the eastern United States and southern Canada valued for its nuts and wood. Butternut's survival is threatened by butternut canker; a disease caused by the exotic fungus Sirococcus clavigignenti-juglandacearum Nair, Kostichka & Kuntz. Japanese walnut (J. ailantifolia Carr.) native to Japan and Sakhalin, which was introduced into North America from Japan about 1870 by nurserymen can also serve as a host to S. clavigignenti-juglandacearum (Scj). Hybrids between butternut and Japanese walnut (J.×bixbyi but commonly called buartnuts or just buarts) are difficult to distinguish morphologically from butternuts, and scientists have expressed concern over the possibility of range-wide genetic invasion by Japanese walnut via hybridization with butternut. Black walnut (J. nigra L.) is a large tree native throughout the eastern United States from New England to Texas. It is a valuable hardwood species that also produces a high quality, edible nut.We used pair-wise combinations of forty random primers to screen bulked DNA pools of butternut, Japanese walnut and buartnuts to identify genomic regions unique to Japanese walnut. About 530 randomly amplified polymorphic DNA (RAPD) panels were examined. We found about thiry-two DNA amplicons present in Japanese walnut and buartnut hybrids but absent in butternut. We cloned and sequenced twenty four amplicons in order to convert them into useful genomic markers. Nine CAPS or SCAR markers were developed successfully from these RPAD markers. The utility of the markers for identifying hybrids was tested and verified using more than 190 genotypes. The markers will be used to identify buartnut hybrids based on the presence of introgressed genomic fragments inherited from Japanese walnut. A battery of 12 microsatellite markers was used to carry out parentage analysis of 328 9-year-old black walnut progeny and 20 parent trees associated with a black walnut progeny test in Indiana, U. S. A. We obtained the phenotypic data for each open-pollinated progeny family and compared it with data for the same families after each seedling's membership in the family was either confirmed or disproved based on genotypic data. The four parameters height, diameter at breast height (DBH), leader (LDR), and quality were measured for each progeny. The genotypic data also permitted the assignment of some progeny into paternal half-sib and full-sib families. The black walnut nut breeding orchard at the University of Missouri contains about 70 accessions used for breeding. The 22 cultivars were characterized for seven phenological descriptors: bud break date (dyas after'Davidson'(BBDAD), first pistillate date (FPLD), first pollen date (FPD), season length (SL), harvest date after'Davidson'(HDAD), mean pistillate date after'Davidson'(MPLDAD), and mean pollen date after'Davidson'(MPDAD). A battery of 10 microsatellite markers was used to carry out cultivar identification and genetic analysis of 285 samples and 22 cultivars (clones). We combined morphological, phenological, and genotype data from ten microsatellites to group individual accessions by cultivar (clone). The main results were as follows:1. Field observations indicate that trees commonly called buartnut [a hybrid of butternut and its close congener Japanese walnut (J. ailantifolia×J. cinerea)] may be more resistant to butternut canker disease fungus (Scj)than either parental species. We confirmed that hybrids currently growing in the landscape typically have a complex genetic history and present features of the parental species in all possible combinations.2. We cloned and sequenced 32 RAPD amplicons apparently present in JA but absent from JC samples. Of these, primers were designed to amplify 24 loci for re-sequencing. We ultimately identified one internal transcribed spacer region (ITS) marker, one chloroplast (TrnT (UAA)–trnF (GAA) region) marker, one mitochondrial marker (3-9), and six nuclear markers (16R-2, 22-5, 14R-1, 39-6, 40-1, and 15R-8). Five markers were co-dominant (22-5, 14R-1, 39-6, 40-1, and 15R-8), and four markers were dominant (ITS, trnT–trnF, 3-9, and 16R-2).3. The molecular markers described here for identifying buarts and other hybrids were robust, simple to use, easy to score in the lab, and they provide a new resource-management tool. The markers are also flexible, and they have already been used to identify non-hybrid trees in National Forests of United States, for establishing seed orchards, and to further butternut resistant breeding and conservation efforts by the USDA Forest Service and cooperating institutions. The utility of the markers for identifying hybrids was tested and verified using 24 Japanes walnuts, 51 hybrids, 111 butternuts, one J. mandshurica, and four black walnuts. J. mandshurica had a phenotype identical to JA at all the loci we tested, and that all JA samples (100 %) had a JA phenotype at all the loci except for one sample (008) at one locus (15R-8). It was 9.8 % of butternuts we tested turned out to be hybrids, 11.7 % of the butternuts had JA cytoplasm (and so they turned out to be hybrids), 90.2 % of the hybrids had JA cytoplasm.4. Using microsatellite markers and quantitative genetic analyses we identified elite genotypes for further genetic improvement or the establishment of production populations. A sequential exclusion procedure was used in an attempt to determine paternity for all progeny. A high level of genetic diversity was revealed among the seedling in the progenty test (Ho=0.802, He=0.778), and the mean number of alleles per locus was 18, locus WGA82 has 35 alleles, and locus AAG001 has 7 alleles.5. Approximately 89% of the offspring were assigned by parentage analysis to their putative female. Based on the female parents'genotype, paternity tests revealed that about 6% of the offsprings could be assigned a male parent. The probability of error in first parent assignment, second parent assignment, parent pair assignment, individual identity and sib identity was < 0.01 % using 12 microsatellite loci. The genotypic data was also could found the assignment of some progeny into paternal half-sib and full-sib families.6. In black walnut we observed high correlations among height, DBH, LDR, and quality. The correlation between height and DBH was 0.88 and highly significant (P≤0.0001). The quality ratings at age nine were strongly and significantly correlated with both height and diameter (r=0.57 and 0.46, respectively, P≤0.0001), but the presence of a central leader (LDR, an important factor in determining if a young hardwood will develop into a crop tree) was only weakly associated with height (r=0.18, P≤0.001) and not significantly associated with diameter. Analyses based on six morphological descriptors of black walnut showed that BBDAD and FPLD were better than other evaluated traits for determing clone identify. The genotypic data showed that the clones used for black walnut nut breeding possessed a highly level of genetic diversity (Ho=0.834, He=0.837), and the mean number alleles per locus was 13, including the locus WAG82 has 20 alleles, locus WGA69 has 7 alleles.7. We found that many black walnut ramets that shared cultivar names did not have identical phenotypes or genotypes, and conversely, some genotypes were given multiple cultivar names. Our results indicate that multiple errors were committed during the propagation of these important cultivars, and that it may be difficult or even impossible to properly assign a name to a genotype.8. Microsatellites provide a powerful and valuable tool for the management of black walnut nut and timber breeding populations. The results showed that genotypic data can be an important supplement to breeding, and that progeny tests and breeding programs are subject to potentially costly errors without genotypic data. |
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