James J. Luby

Phylogenetic Relationships Among Species of Fragaria (Rosaceae) Inferred from Non-coding Nuclear and Chloroplast DNA Sequences

Abstract Phylogenetic relationships among 43 accessions of Fragaria, representing 14 species, and one accession of the outgroup Potentilla fruticosa, were assessed using nucleotide sequence data from the nuclear ITS region and from a region of the chloroplast genome including the trnL intron and the trnL-trnF spacer. Variability was low in both regions and lower in the chloroplast than in the nuclear sequences. The data were analyzed cladistically, separately and in combination. Greater phylogenetic resolution was obtained with the ITS and combined data than with the trn data; the strict consensus tree from the combined data analysis was very similar to that from the ITS analysis, but the position of some accessions was affected by the inclusion of the trn data. The results support the following conclusions: Fragaria iinumae is sister to all other species in the genus; F. vesca and F. nubicola are the diploid species most closely related to the polyploid species, including the tetraploid F. orientalis, the hexaploid F. moschata, and the octoploids F. chiloensis and F. virginiana; the latter two species are sister taxa; accessions of F. virginiana subsp. platypetala from Oregon and Nevada are more closely related to F. chiloensis than to other accessions of F. virginiana. The data provide support for the identification of some accessions in the National Clonal Germplasm Repository in Corvallis, Oregon. Communicating Editor: Kathleen A. Kron

Utilizing wild Fragaria virginiana in strawberry cultivar development: Inheritance of photoperiod sensitivity, fruit size, gender, female fertility and disease resistance

The genetics of photoperiod sensitivity, flowering date, fruit size, gender, female fertility, and disease resistance were investigated in progeny between sets of elite F. virginiana selections and F. × ananassa cultivars and selections planted at sites in Michigan, Minnesota and Ontario. Progeny means varied considerably for all the production traits. Most notable were the large fruit and high fertility observed in crosses with High Falls 22 at all three sites, and Montreal River 10 in Ontario and Michigan. Fragaria virginiana ssp. virginiana parents yielded progeny with much larger fruit than F. virginianassp. glauca parents. General combining ability was significant for all traits at all locations, while specific combining ability was significant for only fruit diameter, ovule set and fruit set in Michigan. Overall, the highest number of day-neutral genotypes were detected in Ontario (mean =44%) compared to Minnesota (31%) and Michigan (26%). In progeny populations of day-neutral F. × ananassa × short-day F. virginiana almost all fit the 1:1 ratio expected if day-neutrality is regulated by a single dominant gene; however, only a few families of short-day F. × ananassa ×day-neutral F. virginianacrosses fit a 1:1 ratio. Likewise, in progeny of day-neutral F. virginiana ×day-neutral F × ananassa crosses, only a few of them fit the 3:1 ratio expected if day-neutrality is regulated by a single dominant gene. These data suggest that it should be relatively easy to useF. virginiana germplasm in strawberry cultivar improvement, and that several different sources of day-neutrality may exist in natural populations.

Morphological and molecular variation among populations of octoploid Fragaria virginiana and F. chiloensis (Rosaceae) from North America.

Relationships among 37 North American octoploid strawberry populations were studied by evaluating 44 morphological traits and 36 randomly amplified polymorphic DNA (RAPD) markers. Both data sets were analyzed by principal components analysis and UPGMA clustering based on genetic distances. Morphological data defined five groups: east of the Missouri River (Fragaria virginiana ssp. virginiana), the Black Hills (F. virginiana ssp. virginiana and ssp. glauca), from the eastern Cascades to the eastern Rocky Mountains (F. virginiana ssp. glauca), the western Cascades and Olympic Peninsula (F. virginiana ssp. platypetala), and the Pacific coast (F. chiloensis). Canonical discriminant analysis clearly discriminated populations into these provenances, suggesting that these groups are morphologically distinct. RAPD data defined three groups, one with F. virginiana ssp. virginiana and ssp. glauca, another with F. chiloensis, and a third with F. virginiana ssp. platypetala. The latter was more similar to F. chiloensis than F. virginiana, suggesting it is likely a subspecies of F. chiloensis. All octoploid North American strawberries have likely derived from a common ancestor and have differentiated into F. chiloensis and F. virginiana by adapting to moister and drier environments, respectively.

Genetic Resources at Our Doorstep: The Wild Strawberries

cessible genetic resources: plants literally at our doorstep. Fortunately, strawberry breeders and related scientists are actively exploiting genes from the wild ancestors of this New World crop to provide stable productivity for farmers and high-quality fruit for consumers. In recent years, germ plasm explorers have systematically collected and evaluated wild strawberries. Genes from these collections are, in turn, being incorporated into breeding populations to control disease and pest problems and afford tolerance to environmental stresses. Only by selection under specific environmental conditions have breed-

A strategy for developing representative germplasm sets for systematic QTL validation, demonstrated for apple, peach, and sweet cherry

Horticultural crop improvement would benefit from a standardized, systematic, and statistically robust procedure for validating quantitative trait loci (QTLs) in germplasm relevant to breeding programs. Here, we describe and demonstrate a strategy for developing reference germplasm sets of perennial, clonally propagated crops, especially those with long juvenile periods. Germplasm is chosen to efficiently represent important members of larger pedigree-connected genepools. To facilitate validation of multiple QTLs, genome-wide representation of alleles is optimized for designated important breeding parents (IBPs) by estimating average allelic representation in relatives. The strategy and arising principles were demonstrated in a simulated germplasm set. Strong statistical power can be achieved with a carefully chosen germplasm set composed of IBPs, their numerous unselected progenies and close relatives, and all available founders and intermediate ancestors. Crop Reference Sets were developed in the marker-assisted breeding (MAB)-enabling “RosBREED” project as a base resource for QTL validation in US breeding germplasm of apple (Malus × domestica), peach (Prunus persica), and sweet cherry (Prunus avium) consisting of 467, 452, and 268 individuals, respectively. These sets adequately represent the most designated IBPs, have distinct advantages for QTL validation over other germplasm arrangements of equal size, and are recommended as a base resource for QTL validation by breeders of these US crops. The strategy described here can be used to develop efficient reference germplasm sets suiting other breeding genepools or to calculate the statistical power for QTL validation of germplasm sets already established.

What data determine whether a plant taxon is distinct enough to merit legal protection? A case study of Sedum integrifolium (Crassulaceae).

Measures of molecular and morphological genetic variation are often used to set conservation priorities and design management strategies for plant taxa. Evaluated together they can give insights into a taxons evolutionary status that neither data type alone can achieve. We investigated the distinctness and variability of Sedum integrifolium ssp. leedyi, a federally and state-listed taxon, from its conspecific relatives using 33 random amplified polymorphic DNA (RAPD) markers (253 plants) and 37 morphological characters from 1308 common-garden-grown plants. We included S. integrifolium ssp. leedyi (four populations), its conspecific relatives (11 populations), and S. rosea and S. rhodanthum populations in our study. The morphological and molecular data correspond in showing that S. integrifolium ssp. leedyi populations are highly distinct. However, the data sets differ in their estimates of the relatedness of some S. integrifolium ssp. leedyi populations and in the percentage variation detected due to differences among them (25 and 9-13% for the molecular and morphological data, respectively) suggesting little gene flow among populations and some differentiation, possibly from selective pressures. Given our data, we recommend that S. integrifolium ssp. leedyi merits protection under the U.S. Endangered Species Act and that its populations be managed as distinct units.

A next-generation marker genotyping platform (AmpSeq) in heterozygous crops: a case study for marker-assisted selection in grapevine

Marker-assisted selection (MAS) is often employed in crop breeding programs to accelerate and enhance cultivar development, via selection during the juvenile phase and parental selection prior to crossing. Next-generation sequencing and its derivative technologies have been used for genome-wide molecular marker discovery. To bridge the gap between marker development and MAS implementation, this study developed a novel practical strategy with a semi-automated pipeline that incorporates trait-associated single nucleotide polymorphism marker discovery, low-cost genotyping through amplicon sequencing (AmpSeq) and decision making. The results document the development of a MAS package derived from genotyping-by-sequencing using three traits (flower sex, disease resistance and acylated anthocyanins) in grapevine breeding. The vast majority of sequence reads (⩾99%) were from the targeted regions. Across 380 individuals and up to 31 amplicons sequenced in each lane of MiSeq data, most amplicons (83 to 87%) had <10% missing data, and read depth had a median of 220–244×. Several strengths of the AmpSeq platform that make this approach of broad interest in diverse crop species include accuracy, flexibility, speed, high-throughput, low-cost and easily automated analysis.

Differences in the apportionment of molecular and morphological variation in North American strawberry and the consequences for genetic resource management

Variation for 24 morphological traits measured in a greenhouse environment and 36 randomly amplified polymorphic DNA (RAPD) markers was assessed among 318 wild octoploid strawberry (Fragariaspp.) genotypes from diverse habitats across the northern USA. RAPD marker frequencies and certain leaf and flower morphology traits (petiole color, leaf mass/area ratio, leaflet length and width, flower and receptacle diameter, petal width, flowers/inflorescence) were significantly different between the F. chiloensis-platypetala and F. virginiana-glauca species complexes. The proportion of variation accounted for by provenance effects was lower for the RAPD markers than for most morphological traits, especially in the F. virginiana-glauca species complex. Morphological traits of potential adaptive importance group the collection into provenances within each species-complex, and reflect the significant habitat and geographic differences across the region from which the germplasm was collected. Variation among populations within provenances was low for the molecular and most morphological traits, with a much larger amount of variability among plants within populations. Most of the variation for the presumably more selectively-neutral RAPD data was among plants within populations and populations within provenances rather than among the provenances that were recognized based on morphological traits, especially in the F. virginiana-glauca complex. Patterns of diversity for morphological traits must be considered, along with more selectively-neutral molecular characters such as RAPDs, to formulate effective sampling strategies and to properly estimate the quantity and apportionment of diversity within this germplasm.

Inheritance of flower color in Dendranthema grandiflora Tzvelev. (Chrysanthemum morifolium Ramat.) using cultivars and inbreds. I: Plastid pigmentation

SummaryProgeny from controlled crosses of 28 garden chrysanthemum (Dendranthema grandiflora Tzvelev.) inbreds in various combinations as males with 3 heterozygous cultivars and 10 inbreds as females were evaluated for plastid pigmentation in a series of field and glasshouse environments. Disomic inheritance of a dominant gene, which acts to inhibit pigmentation, best explained the data. However, 1 of 8 families involving a glasshouse cultivar with colorless plastids (‘Puritan’) as female and inbreds with yellow plastids as males deviated significantly from the expected 1:1 ratio. This family consistently exhibited an excess of plants with colorless plastids. Although parent clones and cloned progeny from inbred parents were scored the same in field and glasshouse environments, reevaluation in the field of all 8 ‘Puritan’ families, first evaluated in a glasshouse environment, revealed phenotypic misclassification apparently due to environmental effects. An excess of plants with colorless plastids appeared in the glasshouse but evaluation of the same genotypes in the field resulted in an excess of plants having yellow plastids. Submerging ray florets in a hot-water bath revealed yellow plastids masked by vacuolar pigments, thus reducing chances of misclassification. The data suggest that evaluation of highly variable families in a single environment can lead to misclassification of plastid pigmentation. However, even with evaluation in 2 or more environments, certain families may exhibit an excess of plants with colorless plastids.

Next Generation Mapping of Enological Traits in an F2 Interspecific Grapevine Hybrid Family.

In winegrapes (Vitis spp.), fruit quality traits such as berry color, total soluble solids content (SS), malic acid content (MA), and yeast assimilable nitrogen (YAN) affect fermentation or wine quality, and are important traits in selecting new hybrid winegrape cultivars. Given the high genetic diversity and heterozygosity of Vitis species and their tendency to exhibit inbreeding depression, linkage map construction and quantitative trait locus (QTL) mapping has relied on F1 families with the use of simple sequence repeat (SSR) and other markers. This study presents the construction of a genetic map by single nucleotide polymorphisms identified through genotyping-by-sequencing (GBS) technology in an F2 mapping family of 424 progeny derived from a cross between the wild species V. riparia Michx. and the interspecific hybrid winegrape cultivar, ‘Seyval’. The resulting map has 1449 markers spanning 2424 cM in genetic length across 19 linkage groups, covering 95% of the genome with an average distance between markers of 1.67 cM. Compared to an SSR map previously developed for this F2 family, these results represent an improved map covering a greater portion of the genome with higher marker density. The accuracy of the map was validated using the well-studied trait berry color. QTL affecting YAN, MA and SS related traits were detected. A joint MA and SS QTL spans a region with candidate genes involved in the malate metabolism pathway. We present an analytical pipeline for calling intercross GBS markers and a high-density linkage map for a large F2 family of the highly heterozygous Vitis genus. This study serves as a model for further genetic investigations of the molecular basis of additional unique characters of North American hybrid wine cultivars and to enhance the breeding process by marker-assisted selection. The GBS protocols for identifying intercross markers developed in this study can be adapted for other heterozygous species.