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- Author or Editor: Brian E. Scheffler x
Primer design is a critical step in the application of PCR-based technologies in genetic diversity analysis of horticultural plants. As more plant genomes have been sequenced in recent years, the emphasis of primer design strategy has shifted to genome-wide and high-throughput direction. This poster summarizes recent advances in primer design for profiling of DNA polymorphism in higher plants, including new primers for the classical plant DNA marker system such as simple sequence repeat (SSR) and single-nucleotide polymorphism (SNPs), as well as newly developed DNA marker systems such as sequence-related amplified polymorphism (SRAP), sequence-specific amplification polymorphism (SSAP), target region amplification polymorphism (TRAP), and universal rice primer (URP). Although most of these primers were designed for agronomical crops, they could be applied to horticultural plants because plant genomes are evolutionarily related. Also, these new primer design strategies could help horticultural researchers develop better primers specifically for profiling of polymorphism in a variety of horticultural crops, invasive weeds, or medicinal plants. We will present examples of their utilization in these diverse systems.
Using 14 codominant microsatellite markers that amplify loci across 14 different Hydrangea L. species, we analyzed gene diversity and genetic similarity within Hydrangea. Samples also included Dichroa Lour., Platycrater Sieb. and Zucc., and Schizophragma Sieb. and Zucc. genera to establish their relatedness to Hydrangea species since previous work suggests they may be closely related. Our results support the close affiliation between Macrophyllae E.M. McClint. and Petalanthe (Maxim.) Rehder subsections and their separation from the other Hydrangea species. Most of the Hydrangea species analyzed cluster within their designated sections and subsections; however, genetic distance between species within each subsection varied considerably. Our data suggest that morphological analyses which labeled H. serrata (Thunb.) Ser. as a subspecies of H. macrophylla (Thunb. Ex J.A. Murr.) Ser. are probably more accurate than recent genome size data suggesting H. macrophylla ssp. macrophylla (Thunb.) Ser. and H. macrophylla ssp. serrata (Thunb.) Makino are separate species. Gene diversity estimates indicate that 64.7% of the total diversity is due to differences between species and 49.7% of the overall variation is due to differences between subsections. Low diversity suggests a lack of gene flow between species and subsections and underscores the difficulty in making wide hybrids. Since only 35.3% of the genetic variation is common to all species, unique alleles were used to develop a molecular key for unambiguous species identification and interspecific hybrid verification. Genetic similarity estimates for all 85 samples suggests a roadmap for introgressing horticulturally important traits from different Hydrangea species.
Recent evidence suggests a close genetic relationship between Hydrangea macrophylla (Thunb.) Ser. and D. febrifuga Lour., which supports previous morphological and DNA sequence data. This relationship was confirmed by the production of fertile intergeneric hybrids. We characterize the genetic diversity of available D. febrifuga plants, both cultivars and wild-collected taxa, as breeding material to improve H. macrophylla. Relatively high genetic diversity is seen among D. febrifuga, which splits into two main clusters. We also document considerable differences in genome size when compared with previously characterized D. febrifuga. Dichroa versicolor (Fortune) D.R. Hunt plants were also included and data suggest that D. versicolor could be a hybrid between H. macrophylla and D. febrifuga, similar to the intergeneric hybrids produced by recent breeding efforts. Because native H. macrophylla plants do not overlap extensively with D. febrifuga populations, we tested Hydrangea indochinensis Merr. as a possible parent because endemic H. indochinensis populations overlap regions where D. febrifuga and D. versicolor have been collected. However, results suggest that H. indochinensis does not share a genetic background with D. versicolor. Taxonomic revision of Dichroa is warranted, especially because we document several more intergeneric hybrids from self-sown, open-pollinated sources.
The genus Chionanthus (Oleaceae Hoffmans. & Link) includes deciduous or evergreen trees and shrubs distributed widely in tropical and sub-tropical areas, including a few temperate species. Although Chionanthus species are planted as ornamental garden plants and commercialized for natural products, genetic information for Chionanthus spp. is lacking. We created microsatellite-enriched libraries of Chionanthus retusus Lindl. & Paxton, assembled 1072 contigs, and detected 1010 repeats. The frequency of the repeats decreased with the increase in repeat length, and the most abundant motifs were: AG, AC, AAG, ACC, AT, and ACTC. We screened 384 markers on 12 accessions of four related taxa that included C. retusus, Chionanthus virginicus L., Chionanthus pygmaeus Small, and Osmanthus americanus (L.) Benth. & Hook. A total of 195 simple sequence repeat (SSR) markers amplified and discriminated six accessions of C. retusus and 57 SSR markers amplified and discriminated across the four Oleaceae species screened. To identify the best markers to use in future experiments, the “Unique Pattern Informative Combination” (UPIC) values were calculated for all the markers and the 100 markers that were most effective are reported here. The percentage of heterozygous loci across the 384 markers was lowest for C. retusus (29.3%) and highest for O. americanus (68.9%). The SSR markers developed here could assist in taxonomy and hybridization investigations for breeding programs and authentication of varieties used as medicinal plants.
Viburnum dilatatum is a popular and economically important ornamental shrub. The wide range of desirable horticultural traits, paired with a propensity for seedlings to become invasive, has created interest in the genetics and breeding of this species. To investigate the genetic diversity of V. dilatatum, microsatellite loci were identified from a GT-enriched genomic library constructed from V. dilatatum ‘Asian Beauty’. Eleven microsatellite loci have been characterized on a group of 16 different related V. dilatatum cultivars and hybrids. Two to 12 alleles were identified per locus, and the polymorphism information content (PIC) values ranged from 0.36 to 0.87. Expected heterozygosity (He) ranged from 0.48 to 0.88 and observed heterozygosity (Ho) ranged from 0 to 0.73. This set of molecular markers also exhibited expected transferability between various V. dilatatum cultivars and two hybrids with V. japonicum. As a consequence, these markers will aid in breeding for new cultivar development, assist with early detection and screening of plants that have escaped cultivation, and are expected to help in refining the phylogenetic relationship of V. dilatatum to other species and genera within the Adoxaceae.
Viburnum rufidulum is a deciduous tree native to North America that has four-season appeal, which provides commercial horticultural value. In addition, the plant has unique and attractive red pubescence on leaf buds and petioles, common to no other Viburnum species. As habitat undergoes development and subsequent fragmentation of native plant populations, it is important to have baseline genetic information for this species. Little is known about the genetic diversity within populations of V. rufidulum. In this study, seven microsatellite loci were used to measure genetic diversity, population structure, and gene flow of 235 V. rufidulum trees collected from 17 locations in Kentucky and Tennessee. The genotype data were used to infer population genetic structure using the program InStruct and to construct an unweighted pair group method with arithmetic mean dendrogram. A single population was indicated by the program InStruct and the dendrogram clustered the locations into two groups; however, little bootstrap support was evident. Observed and expected heterozygosity were 0.49 and 0.78, respectively. Low-to-moderate genetic differentiation (F ST = 0.06) with evidence of gene flow (Nm = 4.82) was observed among 17 populations of V. rufidulum. A significant level of genetic diversity was evident among V. rufidulum populations with most of the genetic variations among individual trees (86.37%) rather than among populations (13.63%), and a Mantel test revealed significant correlation between genetic and geographical distance (r = 0.091, P = 0.001). The microsatellites developed herein provide an initial assessment or a baseline of genetic diversity for V. rufidulum in a limited area of the southeastern region of the United States. The markers are a genetic resource and can be of assistance in breeding programs, germplasm assessment, and future studies of V. rufidulum populations, as this is the first study to provide genetic diversity data for this native species.
Genetic diversity was estimated for 51 Lagerstroemia indica L. cultivars, five Lagerstroemia fauriei Koehne cultivars, and 37 interspecific hybrids using 78 simple sequence repeat (SSR) markers. SSR loci were highly variable among the cultivars, detecting an average of 6.6 alleles (amplicons) per locus. Each locus detected 13.6 genotypes on average. Cluster analysis identified three main groups that consisted of individual cultivars from L. indica, L. fauriei, and their interspecific hybrids. However, only 18.1% of the overall variation was the result of differences between these groups, which may be attributable to pedigree-based breeding strategies that use current cultivars as parents for future selections. Clustering within each group generally reflected breeding pedigrees but was not supported by bootstrap replicates. Low statistical support was likely the result of low genetic diversity estimates, which indicated that only 25.5% of the total allele size variation was attributable to differences between the species L. indica and L. fauriei. Most allele size variation, or 74.5%, was common to L. indica and L. fauriei. Thus, introgression of other Lagestroemia species such as Lagestroemia limii Merr. (L. chekiangensis Cheng), Lagestroemia speciosa (L.) Pers., and Lagestroemia subcostata Koehne may significantly expand crapemyrtle breeding programs. This study verified relationships between existing cultivars and identified potentially untapped sources of germplasm.