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The genetic control of flowering habit in many species of Fragaria has not been well studied. Identification of flowering traits and patterns for these taxa could be used in the quest for perpetual flowering (PF) genes and for the octoploids, broaden the genepool of available PF parents for breeding programs. As such, clones from the Fragaria germplasm collection housed at the USDA-ARS National Clonal Germplasm Repository in Corvallis, OR, were evaluated to describe flowering habits in various taxa and identify PF clones. Flower presence was recorded monthly for 962 clones of 36 taxa from the first of May through October in 2015 and 2016 to determine flowering habit and pairwise comparisons between taxa were examined using Pearson’s Chi-squared test. Taxa with the largest percent of PF accessions were F. vesca subsp. vesca f. semperflorens, F. vesca subsp. vesca f. alba, F. vesca subsp. americana, and F. virginiana subsp. glauca. These taxa had similar flowering habits to each other but were significantly different (α = 0.05) from most other taxa in which the seasonal flowering (SF) trait was predominant. Fifteen clones that demonstrated the PF phenotype in both 2015 and 2016 were identified. Differing genetic controls have been observed for flowering habit in F. ×ananassa and F. vesca. Additional studies are needed to determine genetic control of flowering in other Fragaria taxa.
Development of optimum protocols for micropropagation of C. avellana is particularly important due to the threat of Eastern Filbert Blight and the need for rapid increase of resistant varieties and advanced selections. Therefore, efforts were directed at in vitro establishment, multiplication and rooting, starting with buds from mature trees. The frequency of shoot formation from buds was highest in August but varied with the genotype. Medium containing high Ca levels was more effective in preventing bud necrosis than MS medium. Multiplication rates of 4-7 new shoots/propagule were obtained over a 6-week culture period. Rooting of some genotypes could be accomplished by inclusion of 1 or 3 μM β- indolebutyric acid (IBA) in the medium. Other genotypes responded better to a dip of shoot bases in 1-10 mM IBA for 10 sec., followed by a passage on auxin-free medium. Large numbers of healthy plantlets have been produced for transfer to soil.
The NRT1gene family encodes transport proteins with dual or low affinity for nitrate. The objectives of this experiment were to develop a system that could be used to compare the expression of the NRT1genes between species. This was accomplished by comparing sequences of NRT1homologues from various species and designing degenerate primers in regions of high homology. These primers were used to amplify a region of the NRT1gene from species of interest. A 635 bp PCR product was amplified from each species using the MD2-1 (5' ATGTTACCAAYWTGGGCMAC-3') and MD2-2 (5'-GCCAMWARCCARTAGAAAT-3') primers. The PCR products were cloned and sequenced. At the nucleotide level, CornussericeaL. `Kelseyi' and RhododendronL. `Unique' were 79.52% identical. Species-specific primers were designed and used for RT-PCR to compare NRT1expression in roots of hydroponically grown C. sericea, C. sericea `Kelseyi', and Rhododendron`Unique'. The relative levels of NRT1expression, normalized using 18S rRNA as a standard, were ≈3.2 to 1.7 to 1.0 for C. sericea, C. sericea `Kelseyi', and Rhododendron`Unique', respectively. This approach may eventually be used to examine nitrate uptake potential in different taxa of plants at different times during the growing season.
Hazelnut (Corylus avellana L.) softwood cuttings of the cultivars Ennis and Casina were propagated under mist during June and July 1987 and 1988. Rooting of stem cuttings was stimulated by both Agrobacterium and IBA treatment; however, IBA caused nearly complete bud abscission. Better rooting and bud retention were observed in `Casina' than in `Ennis' in 1988. Bud retention on Agrobacterium -inoculated cuttings improved as the cuttings approached the semi-hardwood stage. Six months after transplanting, Agrobacterium -inoculated hazelnut cuttings had an extensive root system, characteristic of hairy root. Although the mechanism remains unclear, strains of Agrobacterium rhizogenes are effective rooting agents in hazelnut and may cause less bud abscission than IBA. Chemical name used: 1 H -indole-3-butyric acid (IBA).
The use of interspecific hybridization in blueberry (Vaccinium L. section Cyanococcus Gray) breeding has resulted in the incorporation of novel traits from wild germplasm and the expansion of the geographic limits of highbush blueberry (V. ×corymbosum L.) production. The objectives of this study were: 1) to estimate the impact of wide hybridization on inbreeding, heterozygosity, and coancestry of the cultivated tetraploid highbush blueberry; 2) to establish the usefulness of microsatellite markers in assessing genetic relationships among southern highbush blueberry [SHB (V. ×corymbosum)] cultivars; and 3) to report on the expected genetic contribution of wild Vaccinium clones to SHB cultivars. Pedigrees of 107 highbush blueberry cultivars were used to calculate tetrasomic inbreeding coefficients (F), pedigree-based genetic distances, and expected genetic contributions of wild clones. Genotyping data from 21 single-locus microsatellite markers screened across 68 genotypes were used to calculate heterozygosity and proportion of shared alleles distances (Dsa). The results indicated that the effects of wide hybridization on genetic diversity of cultivated blueberry were lower than previously thought. First, SHB cultivars exhibited similar levels of molecular relatedness as historical northern highbush blueberry [NHB (V. ×corymbosum)] cultivars (median Dsa between pairs of cultivars equals 0.19 in both cultivar groups), despite the broader genetic base and larger pedigree distances in the former cultivar group. Second, levels of heterozygosity in modern NHB cultivars were not statistically different from those of SHB (χ2 = 4.0; df = 3; P > 0.05), despite the considerably higher levels of inbreeding in the former cultivar group (median F equal to 0.0035 and 0.0013, respectively). Furthermore, pedigree-based genetic distances were significantly correlated with Dsa (r = 0.57, P ≤ 0.0001), indicating that microsatellite markers are reliable tools in most cases to assess the genetic relationships among SHB cultivars. Finally, seven Vaccinium species constitute the current genetic base of cultivated SHB. In this article, we report on the expected genetic contributions of V. angustifolium Ait., V. constablaei Gray, V. corymbosum, V. darrowii Camp, V. elliottii Chapman, V. tenellum Ait., and V. virgatum Ait. (syn. V. ashei Reade) clones to 38 SHB cultivars.
Peonies (Paeonia), the grand garden perennial of spring and early summer, are economically important to the international cut flower market. Herbaceous peonies (Paeonia section Paeonia), tree peonies (Paeonia section Moutan), and intersectional crosses between the two types (Itoh Paeonia hybrids) are of interest to gardeners, growers, and nursery producers. Thousands of peony cultivars exist and identity is traditionally determined by experienced horticulturists knowledgeable in plant and bloom characteristics. With DNA extraction possible during any time of the year, molecular markers can provide genotype identity confirmation for dormant roots or mature post-bloom plants. The primary objective of our research was to rapidly and inexpensively develop microsatellite markers in a range of Paeonia species using barcoded Illumina libraries. A secondary objective was to apply these simple sequence repeat (SSR) markers to fingerprint 93 accessions that include tree, intersectional, and herbaceous peonies. We used 21 primers to distinguish cultivars and their close relatives. Also from our sequence information, greater than 9000 primers were designed and are made available.
The advent of next-generation, or massively parallel sequencing technologies has been a boon to the cost-effective development of molecular markers, particularly in nonmodel species. Here, we demonstrate the efficiency of microsatellite or simple sequence repeat (SSR) marker development from short-read sequences in black and red raspberry (Rubus occidentalis L. and R. idaeus L., respectively), compare transferability of markers across species, and test whether the rate of polymorphism in the recovered markers can be improved upon by how marker sequences are chosen. From 28,536,412 black raspberry reads and 27,430,159 reads in red raspberry, we identified more than 6000 SSR sequences in each species and selected 288 of these (144 from each species), for testing in black and red raspberry. A total of 166 SSR primer pairs were identified with informative polymorphism in one or both species. SSRs selected based on different percentages (90% to 97% as compared with ≥98%) of read cluster similarity did not differ in polymorphism rates from each other or from those originating from singletons. Efficiency of polymorphic SSR recovery was nearly twice as high in black raspberry from black raspberry-derived sequences as from red raspberry-derived sequences, while efficiency of polymorphic SSR recovery in red raspberry was unaffected by the source of the primer sequences. Development of SSR markers that are transferable between red and black raspberry for marker-assisted selection, evaluation of genome collinearity and to facilitate comparative studies in Rubus L. will be more efficient using SSR markers developed from black raspberry sequences.
Confirming parentage and clonal identity is an important aspect of breeding and managing germplasm collections of clonally propagated, outcrossing crops, like blackberry (Rubus subgenus Rubus). DNA fingerprinting sets are used to identify off-cross progeny and confirm clonal identity. Previously, a six-simple sequence repeat (6-SSR) fingerprinting set was developed for blackberry using a small number of samples. The usefulness of the 6-SSR fingerprinting set for pedigree confirmation had not been evaluated. Therefore, it was used in this study to validate parentage for 6 and 12 biparental populations from the University of Arkansas (UA) and US Department of Agriculture Agricultural Research Service (USDA-ARS), Horticultural Crops Research Unit (HCRU) breeding programs, respectively. Twenty-seven of the 489 individuals in these breeding populations were identified as off-cross. The 6-SSR fingerprinting set was sufficient for parentage confirmation; however, a total of 61 plants distributed across 28 sets of genotypes could not be distinguished from each other. An 8-SSR fingerprinting set with improved resolution was subsequently developed and used to evaluate 177 Rubus accessions from the USDA-ARS National Clonal Germplasm Repository, UA, and USDA-ARS HCRU programs. The 8-SSR fingerprinting set distinguished all samples expected to have unique genotypes and identified differing DNA fingerprints for two sets of accessions suspected to have identical fingerprints. Cluster analysis grouped the accessions from the eastern and western US breeding programs based on geography and descent. Future work will focus on establishing a database of DNA fingerprints for germplasm identification and for determining pedigree relationships between blackberry accessions.
Edible european pears (Pyrus communis L. ssp. communis) are derived from wild relatives native to the Caucasus Mountain region and eastern Europe. Microsatellite markers (13 loci) were used to determine the relationships among 145 wild and cultivated individuals of P. communis maintained in the National Plant Germplasm System (NPGS). A Bayesian clustering method grouped the individual pear genotypes into 12 clusters. Pyrus communis ssp. caucasica (Fed.) Browicz, native to the Caucasus Mountains of Russia, Crimea, and Armenia, can be genetically differentiated from P. communis ssp. pyraster L. native to eastern European countries. The domesticated pears cluster closely together and are most closely related to a group of genotypes that are intermediate to the P. communis ssp. pyraster and the P. communis ssp. caucasica groups. Based on the high number of unique alleles and heterozygosity in each of the 12 clusters, we conclude that genetic diversity of wild P. communis is not fully represented at the NPGS. Additional diversity may be present in seed accessions stored in the NPGS and more pear diversity could be captured through supplementary collection trips to eastern Europe, the Caucasus Mountains, and the surrounding countries.
The availability of strawberry (Fragaria ×ananassa) genomic resources has increased dramatically in recent years. Some of these resources are readily applicable to strawberry breeding programs for use in DNA-informed breeding. Information about these tests and how to interpret them is dispersed through numerous manuscripts or in the laboratories that use them routinely. To assist breeders in identifying tests available to their breeding program and in implementing them in their program, a compendium of strawberry DNA tests was created. This compendium is available for download from the Genome Database for Rosaceae (https://www.rosaceae.org/organism/Fragaria/x-ananassa?pane=resource-4). This resource will be updated continually as old tests are modified and new tests are created.