Morphological traits were examined in an F3 generation derived from a cross between C. lanatus var. lanatus [(Thunb.) Matsum. & Nakai] and C. lanatus var. citroides. At least three genes, C (yellow) vs. c (red), i (inhibitory to C) vs. I (non-inhibitory to C), and y (yellow) vs. yw (white), with epistatic and inhibitory actions were found to govern the inheritance of fruit flesh color. The high frequency of yellow-fleshed fruit and low frequencies of white and red fruits can be explained by the presence of a new allele (yw recessive to y) in the multiple allele series at the Y locus. The low frequency of tan colored seeds in segregating populations could be explained by at least three genes governing inheritance of seed-coat color. Single factor analysis of variance was conducted for each pairwise combination of random amplified polymorphic DNA (RAPD) locus and fruit or seed characteristics. Several RAPD loci were identified to be loosely linked to morphological characteristics.
Leigh K. Hawkins, Fenny Dane, and Thomas L. Kubisiak
Ping Lang, Fenny Dane, Thomas L. Kubisiak, and Hongwen Huang
The genus Castanea (Fagaceae), which contains three sections and seven species, is widely distributed in the deciduous forests of the Northern Hemisphere. The phylogeny of Castanea was estimated using DNA sequence data from five different regions of the chloroplast genome. Sequencing results support the genus Castanea as a paraphyletic group with C. crenata, the Japanese chestnut, representing an early divergence in the genus. The three Chinese species form a strongly supported sister clade to the North American and European clade. A unique westward expansion of extant Castanea species is hypothesized with Castanea originating in eastern Asia, an initial diversification within Asia during the Eocene, followed by intercontinental dispersion and divergence between the Chinese and European/North American species during the Oligocene and a split between the European and North American species in the early Miocene. The differentiation within North America and China might have occurred in late Miocene or early Pliocence. The North America species are supported as a clade with C. pumila var. ozarkensis, the Ozark chinkapin, as the basal lineage, sister to the group comprising C. pumila var. pumila, the Allegheny chinkapin, and C. dentata, the American chestnut. Morphological evolution of one nut per bur in the genus may have occurred independently on two continents.
Hongwen Huang, Desmond R. Layne, and Thomas L. Kubisiak
Twelve, 10-base primers amplified a total of 20 intense and easily scorable polymorphic bands in an interspecific cross of PPF1-5 pawpaw [Asimina triloba (L.) Dunal.] × RET (Asimina reticulata Shuttlew.). In this cross, all bands scored were present in, and inherited from, the A. triloba parent PPF1-5. Nineteen of the 20 bands were found to segregate as expected (1:1 or 3:1) based on chi-square goodness-of-fit tests, and were subsequently used to evaluate genetic diversity in populations of A. triloba collected from six states (Georgia, Illinois, Indiana, Maryland, New York, and West Virginia) within its natural range. Analysis of genetic diversity of the populations revealed that the mean number of alleles per locus was A = 1.64, percent polymorphic loci was P = 64, and expected heterozygosity was He = 0.25. No significant differences were found among populations for any of the polymorphic indices. Partitioning of the population genetic diversity showed that the average genetic diversity within populations was Hs = 0.26, accounting for 72% of the total genetic diversity. Genetic diversity among populations was Dst = 0.10, accounting for 28% of the total genetic diversity. Nei's genetic identity and distance showed a high mean identity of 0.86 between populations. Genetic relationships among the populations examined by unweighted pair-group mean clustering analysis separated the six populations into two primary clusters: one composed of Georgia, Maryland, and New York, and the other composed of Illinois, Indiana, and West Virginia. The Georgia and Indiana populations were further separated from the other populations within each group. This study provides additional evidence that marginal populations within the natural range of A. triloba should be included in future collection efforts to capture most of the rare and local alleles responsible for this differentiation.
Leigh K. Hawkins, Fenny Dane, Thomas L. Kubisiak, and Billy Rhodes
Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum f.sp. niveum (FON), is a serious disease of the watermelon (Citrullus lanatus). Three races of this pathogen (races 0, 1, and 2) have been identified based on differential pathogenicity assays. Most commercially available cultivars are resistant to races 0 and 1. Inheritance for resistance to these races is thought to be controlled by a single dominant gene. No cultivars are resistant to race 2 and resistance is thought to be a quantitative trait. F2 lines derived from a cross between the Fusarium-resistant Citrullus lanatus PI296341, and the Fusarium-susceptible watermelon cultivar `New Hampshire Midget' were used to generate a RAPD-based map of the Citrullus genome. F2:3 families were assayed in the greenhouse for resistance to races 1 and 2. Those families that were either highly resistant or highly susceptible were used in identifying markers linked to Fusarium wilt resistance. A preliminary map of the Citrullus genome based on random amplified polymorphic DNA (RAPD) markers has been expanded with the inclusion of simple sequence repeats (SSRs), amplified fragment length polymorphisms (AFLPs), and isozymes.
Hongwen Huang, Desmond R. Layne, and Thomas L. Kubisiak
Kentucky State Univ. (KSU) is the national clonal germplasm repository for Asimina species. Previous evaluation of the KSU pawpaw collection using 24 isozyme markers demonstrated that pawpaw has a relatively higher genetic diversity than that noted for other plant species with similar species characteristics (long-lived, woody, perennial, out-crossing, temperate, widespread, etc.). Current evaluation using RAPD markers will provide us with a more-accurate insight into pawpaw genetic diversity and population structure. In a preliminary experiment, one hundred 10-mer primers (OA1-20 through OE1-20, Operon Technologies Inc.) were screened against 32 commercial cultivars or advanced selections. A subset of 24 primers that amplify only the most-informative markers were used for germplasm evaluation. Sixty-eight RAPD markers were identified and used for determining genetic parameters. One-hundred-twenty pawpaw accessions were sampled from the KSU repository for RAPD analysis. These accessions represented nine widely distributed states within pawpaw's native range. RAPD data were subjected to various analyses using the NTSYS-PC computer program (ver. 1.8). Information generated from isozyme and RAPD markers will be used to formulate future germplasm collection strategies from wild populations within the native range. The implications of such information to the genetic enhancement of our repository and establishment of a core collection will be discussed.
Hongwen Huang, Desmond R. Layne, and Thomas L. Kubisiak
Thirty-four extant pawpaw [Asimina triloba (L.) Dunal] cultivars and advanced selections representing a large portion of the gene pool of cultivated pawpaws were investigated using 71 randomly amplified polymorphic DNA (RAPD) markers to establish genetic identities and evaluate genetic relatedness. All 34 cultivated pawpaws were uniquely identified by as few as 14 loci of eight primers. Genetic diversity of the existing gene pool of cultivated pawpaws, as estimated by Nei's gene diversity (He), was similar to that of wild pawpaw populations. The genetic relatedness among the cultivated pawpaws examined by UPGMA cluster analysis separated 34 cultivars and selections into two distinct clusters, a cluster of PPF (The PawPaw Foundation) selections and a cluster including a majority of the extant cultivars selected from the wild and their derived selections. The results are in general agreement with the known selection history and pedigree information available. The consensus fingerprint profile using the genetically defined RAPD markers is a useful and reliable method for establishing the genetic identities of the pawpaw cultivars and advanced selections. This also proved to be an improved discriminating tool over isozyme markers for the assessment of genetic diversity and relatedness. RAPD profiling of data presented in this study provides a useful reference for germplasm curators engaged in making decisions of sampling strategies, germplasm management and for breeders deciding which parents to select for future breeding efforts.
Leigh K. Hawkins, Fenny Dane, Thomas L. Kubisiak, Billy B. Rhodes, and Robert L. Jarret
Isozyme, randomly amplified polymorphic DNA (RAPD), and simple sequence repeats (SSR) markers were used to generate a linkage map in an F2 and F3 watermelon [Citrullus lanatus (Thumb.) Matsum. & Nakai] population derived from a cross between the fusarium wilt (Fusarium oxysporum f. sp. niveum) susceptible `New Hampshire Midget' and resistant PI 296341-FR. A 112.9 cM RAPD-based map consisting of 26 markers spanning two linkage groups was generated with F2 data. With F3 data, a 139 cM RAPD-based map consisting of 13 markers covering five linkage groups was constructed. Isozyme and SSR markers were unlinked. About 40% to 48% of the RAPD markers were significantly skewed from expected Mendelian segregation ratios in both generations. Bulked segregant analysis and single-factor analysis of variance were employed to identify RAPD markers linked to fusarium wilt caused by races 1 and 2 of F. oxysporum f. sp. niveum. Current linkage estimates between the resistance trait and the marker loci were too large for effective use in a marker-assisted selection program.
Hongwen Huang, Zuozhou Li, Jianqiang Li, Thomas L. Kubisiak, and Desmond R. Layne
Phylogenetic relationships within the Actinidia were investigated using randomly amplified polymorphic DNA (RAPD) markers. DNAs from 40 taxa, including 31 species encompassing all four sections and four series of the traditional subdivisions within the genus, were amplified using 22 preselected 10-mer oligonucleotide primers. A total 204 DNA bands were scored across the 40 taxa, of which 188 (92%) were polymorphic. A wide range of genetic similarity was observed among the taxa (0.13 to 0.61). The average similarity between varieties of the same species was 0.54, and between different species was 0.28, respectively. Although the phylogenetic analysis revealed a clear indication that section Leiocarpae was a monophyletic group, subdivisions of the other three traditional sections were poorly supported. The UPGMA phenogram showed that the majority of the species clustered into geographic subgroups in accordance with their natural distribution (the Yangtzi River, southeastern China, southern China and southwestern China). The intrageneric subdivisions of Actinidia appeared to be difficult, but some subdivisions could be explained by the geographic distribution of the species, particularly for species of Liang's sections of Maculatae and Stellatae. The phylogenetic relationships among several species with previous taxonomic uncertainty are also discussed on the basis of the RAPD data. The results of this study supplement our previous understanding of the Actinidia taxonomy based solely on morphological characters.