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Quinoa is a potential new crop for New England; however, its susceptibility to downy mildew, caused by Peronospora variabilis, is a key obstacle for cultivation. The objectives of this study were to evaluate differential resistance within the Chenopodium genus, identify novel sources of resistance for use in future genetic studies or breeding programs, and investigate phylogenetic relationships of P. variabilis isolates from different Chenopodium hosts. The long-term goal of this research is to develop a resistant variety of quinoa to be grown in New England. Field trials conducted at the University of New Hampshire evaluated downy mildew disease severity on 10 Chenopodium accessions representing four species. Disease severity for each treatment was compared and significant differences in disease severity were observed between accessions. C. berlandieri var. macrocalycium ecotypes collected from Rye Beach, New Hampshire and Appledore Island, Maine exhibited the lowest disease severity over the growing season. P. variabilis was isolated from each accession, and COX2 sequences were compared. Phylogenetic analyses suggest no effect of host species on P. variabilis sequence similarity; however, isolates are shown to cluster by geographic location. This research provides the first step in identifying potential New England native sources of resistance to downy mildew within the genus Chenopodium and provides preliminary information needed to further investigate resistance at the genomic level in Chenopodium spp.

Quinoa is a potential new crop for New England; however, its susceptibility to downy mildew, caused by

Background Like most legumes, chickpeas form specialized organs called root nodules. These nodules allow for a symbiotic relationship with rhizobium bacteria. The rhizobia provide fixed atmospheric nitrogen to the plant in a usable form. It is of both basic and practical interest to understand the host plant genetics of legume root nodulation. Chickpea lines PM233 and PM405, which harbor the mutationally identified nodulation genes rn1 and rn4, respectively, both display nodulation-deficient phenotypes. Previous investigators identified the rn1 mutation with the chickpea homolog of Medicago truncatula nodulation gene NSP2, but were unable to define the mutant rn1 allele. We used Illumina and Nanopore sequencing reads to attempt to identify and characterize candidate mutation sites responsible for the PM233 and PM405 phenotypes. Results We aligned Illumina reads to the available desi chickpea reference genome, and did a de novo contig assembly of Nanopore reads. In mutant PM233, the Nanopore contigs allowed us to identify the breakpoints of a ~ 35 kb deleted region containing the CaNSP2 gene, the Medicago truncatula homolog of which is involved in nodulation. In mutant PM405, we performed variant calling in read alignments and identified 10 candidate mutations. Genotyping of a segregating progeny population narrowed that pool down to a single candidate gene which displayed homology to M. truncatula nodulation gene NIN. Conclusions We have characterized the nodulation mutation sites in chickpea mutants PM233 and PM405. In mutant PM233, the rn1 mutation was shown to be due to deletion of the entire CaNSP2 nodulation gene, while in mutant PM405 the rn4 mutation was due to a single base deletion resulting in a frameshift mutation between the predicted RWP-RK and PB1 domains of the NIN nodulation gene. Critical to characterization of the rn1 allele was the generation of Nanopore contigs for mutant PM233 and its wild type parent ICC 640, without which the deletional boundaries could not be defined. Our results suggest that efforts of prior investigators were hampered by genomic misassemblies in the CaNSP2 region of both the desi and kabuli reference genomes.

The Rosaceae crop family (including almond, apple, apricot, blackberry, peach, pear, plum, raspberry, rose, strawberry, sweet cherry, and sour cherry) provides vital contributions to human well-being and is economically significant across the U.S. In 2003, industry stakeholder initiatives prioritized the utilization of genomics, genetics, and breeding to develop new cultivars exhibiting both disease resistance and superior horticultural quality. However, rosaceous crop breeders lacked certain knowledge and tools to fully implement DNA-informed breeding—a “chasm” existed between existing genomics and genetic information and the application of this knowledge in breeding. The RosBREED project (“Ros” signifying a Rosaceae genomics, genetics, and breeding community initiative, and “BREED”, indicating the core focus on breeding programs), addressed this challenge through a comprehensive and coordinated 10-year effort funded by the USDA-NIFA Specialty Crop Research Initiative. RosBREED was designed to enable the routine application of modern genomics and genetics technologies in U.S. rosaceous crop breeding programs, thereby enhancing their efficiency and effectiveness in delivering cultivars with producer-required disease resistances and market-essential horticultural quality. This review presents a synopsis of the approach, deliverables, and impacts of RosBREED, highlighting synergistic global collaborations and future needs. Enabling technologies and tools developed are described, including genome-wide scanning platforms and DNA diagnostic tests. Examples of DNA-informed breeding use by project participants are presented for all breeding stages, including pre-breeding for disease resistance, parental and seedling selection, and elite selection advancement. The chasm is now bridged, accelerating rosaceous crop genetic improvement.

The first high-resolution genetic linkage map of the ancestral octoploid (2n = 8x = 56) strawberry species, Fragaria virginiana, was constructed using segregation data obtained from a pentaploid progeny population. This novel mapping population of size 178 was generated by crossing highly heterozygous F. virginiana hybrid “LB48” as a paternal parent with diploid (2n = 2x = 14) Fragaria vesca “Hawaii 4”. The LB48 linkage map comprises 6055 markers genotyped on the Axiom® IStraw90 strawberry SNP array. The map consists of 28 linkage groups (LGs) organized into seven homoeology groups of four LGs each, and excludes a small 29th LG of undefined homoeology. One member of each homoeology group was assignable to an “A” subgenome associated with ancestral diploid Fragaria vesca, while no other subgenomes were defined. Despite an intriguing discrepancy within homoeology group VI, synteny comparisons with the previously published Fragaria ×ananassa DA × MO linkage map revealed substantial agreement. Following initial map construction, examination of crossover distributions revealed that six of the total 5162 (=29 chromosomes/individual × 178 individuals) chromosomes making up the data set exhibited abnormally high crossover counts, ranging from 15 to 48 crossovers per chromosome, as compared with the overall mean of 0.66 crossovers per chromosome. Each of these six hyper-recombinant (HypR) chromosomes occurred in a different LG and in a different individual. When calculated upon exclusion of the six HypR chromosomes, the canonical (i.e., broadly representative) LB48 map had 1851 loci distributed over a total map length of 1873 cM, while their inclusion increased the number of loci by 130, and the overall map length by 91 cM. Discovery of these hyper-recombinant chromosomes points to the existence of a sporadically acting mechanism that, if identified and manipulable, could be usefully harnessed for multiple purposes by geneticists and breeders.

The subgenomic compositions of the octoploid (2n = 8× = 56) strawberry (Fragaria) species, including the economically important cultivated species Fragaria x ananassa, have been a topic of long-standing interest. Phylogenomic approaches utilizing next-generation sequencing technologies offer a new window into species relationships and the subgenomic compositions of polyploids. We have conducted a large-scale phylogenetic analysis of Fragaria (strawberry) species using the Fluidigm Access Array system and 454 sequencing platform. About 24 single-copy or low-copy nuclear genes distributed across the genome were amplified and sequenced from 96 genomic DNA samples representing 16 Fragaria species from diploid (2×) to decaploid (10×), including the most extensive sampling of octoploid taxa yet reported. Individual gene trees were constructed by different tree-building methods. Mosaic genomic structures of diploid Fragaria species consisting of sequences at different phylogenetic positions were observed. Our findings support the presence in octoploid species of genetic signatures from at least five diploid ancestors (F. vesca, F. iinumae, F. bucharica, F. viridis, and at least one additional allele contributor of unknown identity), and questions the extent to which distinct subgenomes are preserved over evolutionary time in the allopolyploid Fragaria species. In addition, our data support divergence between the two wild octoploid species, F. virginiana and F. chiloensis.

The northern New England region includes the states of Vermont, New Hampshire, and Maine and encompasses a large degree of climate and edaphic variation across a relatively small spatial area, making it ideal for studying climate change impacts on agricultural weed communities. We sampled weed seedbanks and measured soil physical and chemical characteristics on 77 organic farms across the region and analyzed the relationships between weed community parameters and select geographic, climatic, and edaphic variables using multivariate procedures. Temperature-related variables (latitude, longitude, mean maximum and minimum temperature) were the strongest and most consistent correlates with weed seedbank composition. Edaphic variables were, for the most part, relatively weaker and inconsistent correlates with weed seedbanks. Our analyses also indicate that a number of agriculturally important weed species are associated with specific U.S. Department of Agriculture plant hardiness zones, implying that future changes in climate factors that result in geographic shifts in these zones will likely be accompanied by changes in the composition of weed communities and therefore new management challenges for farmers.

Background Fragaria vesca or ‘woodland strawberry’ has emerged as an attractive model for the study of ripening of non-climacteric fruit. It has several advantages, such as its small genome and its diploidy. The recent availability of the complete sequence of its genome opens the possibility for further analysis and its use as a reference species. Fruit softening is a physiological event and involves many biochemical changes that take place at the final stages of fruit development; among them, the remodeling of cell walls by the action of a set of enzymes. Xyloglucan endotransglycosylase/hydrolase (XTH) is a cell wall-associated enzyme, which is encoded by a multigene family. Its action modifies the structure of xyloglucans, a diverse group of polysaccharides that crosslink with cellulose microfibrills, affecting therefore the functional structure of the cell wall. The aim of this work is to identify the XTH-encoding genes present in F. vesca and to determine its transcription level in ripening fruit. Results The search resulted in identification of 26 XTH-encoding genes named as FvXTHs. Genetic structure and phylogenetic analyses were performed allowing the classification of FvXTH genes into three phylogenetic groups: 17 in group I/II, 2 in group IIIA and 4 in group IIIB. Two sequences were included into the ancestral group. Through a comparative analysis, characteristic structural protein domains were found in FvXTH protein sequences. In complement, expression analyses of FvXTHs by qPCR were performed in fruit at different developmental and ripening stages, as well as, in other tissues. The results showed a diverse expression pattern of FvXTHs in several tissues, although most of them are highly expressed in roots. Their expression patterns are not related to their respective phylogenetic groups. In addition, most FvXTHs are expressed in ripe fruit, and interestingly, some of them (FvXTH 18 and 20, belonging to phylogenic group I/II, and FvXTH 25 and 26 to group IIIB) display an increasing expression pattern as the fruit ripens. Conclusion A discrete group of FvXTHs (18, 20, 25 and 26) increases their expression during softening of F. vesca fruit, and could take part in cell wall remodeling required for softening in collaboration with other cell wall degrading enzymes. Electronic supplementary material The online version of this article (10.1186/s12864-017-4255-8) contains supplementary material, which is available to authorized users.

BACKGROUND: The fungal disease verticillium wilt has been recognized as an obstacle to strawberry production since its initial description in 1931. The full potential of genetic resistance as a solution to this problem has yet to be determined or realized. OBJECTIVE: Our investigations are concerned with defining new sources of resistance to verticillium wilt disease in cultivated and wild strawberry germplasm, and with advancing genetic studies on the basis of resistance/susceptibility. METHODS: We screened 23 diploid, 1 decaploid, and 26 octoploid Fragaria (strawberry) germplasm accessions and cultigens for response to root-dip inoculation with Verticillium dahliae isolate V1. Pedigree relationships of 10 studied cultigens were examined. Crosses were performed between resistant and susceptible accessions. RESULTS: Variability in inoculation response existed within and between species at diploid and octoploid levels. Very or moderately resistant accessions were found within each of three diploid and three octoploid species. Moderately or very susceptible accessions were documented within F. vesca and each octoploid species. Segregation for resistance/susceptibility was evident in progeny populations. CONCLUSIONS: The verticillium wilt resistance ratings reported here and discussed in relation to prior studies adds to the body of publically available knowledge about sources of wilt resistance and susceptibility in Fragaria germplasm.

Ten seedlings from 36 crosses representing eastern and western North American short day and remontant genotypes were evaluated in 2011 and 2012 in California, Michigan, New Hampshire and Oregon, for phenology, flower related traits, plant characteristics, fruit characteristics and fruit chemistry traits. There was significant variability among genotypes, locations and evaluation year for most of the characteristics; however, few genotype × location and genotype × year interactions were detected. General combining ability variance components were significant for all traits and greater than SCA variance components for peduncle length, total flowering weeks, flowering cycles, truss size, growing degree days for harvest data, remontancy, achene position, ease of capping, fruit weight, percent soluble solids, titratable acidity and soluble solids/titratable acidity. ‘Sarian’ was identified as the best contributing parent for remontancy. Narrow-sense heritability estimates were moderate to high (0.33–0.78) for total flowering weeks, flowering cycle, truss size, remontancy, number of runners, fruit weight, pH, and titratable acidity. Having a better understanding of these attributes will provide breeders guidance on the most effective breeding strategies for incorporating superior traits from this germplasm into their programs.

Asian germplasm has significantly contributed to berry crops in America in several ways. The American wild octoploid species [Fragaria chiloensis (L.) Mill. and F. virginiana Mill.], and subsequently, the cultivated strawberry (F. 3ananassa Duch. ex Rozier), have benefitted from Asian heritage in the evolutionary time scale. Second, breeders have combined Asian germplasm in crosses for improved fruit cultivars. Third, Asian temperate fruit species have been collected from wild stands in their native ranges, imported, and in some cases improved and are now cultivated in the West or throughout the world. The objectives of this article were to 1) describe evolutionary contributions of Asian species to the American strawberry genome; 2) present examples of breeding Asian species (Rubus L. subgenus Idaeobatus) into cultivated raspberries; and 3) give examples of two Asian fruit species that have been recently introduced and cultivated or that could be developed for cultivation in the United States. Berries are widely appreciated by the Western palate and have provided highly nutritive components to the human diet for millennia (Hummer and Janick, 2007). Strawberries are a noted American crop, yet they are not without influence from Asian genes. European and American raspberry (Rubus L. subgenus Idaeobatus) cultivars have benefitted greatly from Asian heritage by plant improvement through breeding. In addition, Asian berry species harvested by native peoples from wild stands have potential for cultivation in America. This article summarizes examples of Asian influence in American berry crops. In the strawberry genus, Fragaria L., different ploidy levels are endemic in separate regions or continents (Staudt, 2009). Diploid (2n = 2x = 14) species are distributed in the northern hemisphere; tetraploids are found in Asia; hexaploids in Europe; and octoploids in the Americas (Staudt, 1999, 2009). The two known decaploid species have limited distributions: F. cascadensis Hummer in the Cascade Mountains, OR (Hummer et al., 2012), and F. iturupensis Staudt on Atsunupuri Volcano, Iturup Island, Russian Federation (Hummer et al., 2009). The principal strawberry of commerce is Fragaria ·ananassa. This species is octoploid and originated as an accidental cross between the white-fruited South American F. chiloensis subsp. chiloensis f. chiloensis and the North American F. virginiana subsp. virginiana (Hummer et al., 2011; Staudt, 1999). Scientists have speculated about the evolutionary origins of the American octoploids (Davis et al., 2009). Octoploid taxa have complex genomes, diverse morphologies, and variable, closely related subspecies and forma. Multiple hypotheses concerning the derivation of the octoploids have been proposed, involving allopolyploidy and autopolyploidy (Darrow, 1966; Fedorova, 1946; Folta and Davis, 2006; Senanayake and Bringhurst 1967). Molecular genomic analyses have provided new insight of relevance to this question.

UMR 1332 - Equipe OrFE; The Axiom® IStraw90 SNP (single nucleotide polymorphism) array was developed to enable high-throughput genotyping in allo-octoploid cultivated strawberry (Fragaria ×ananassa). However, high cost ($ 80-105 sample-1) limits throughput for certain applications. On average the IStraw90 has yielded 50 to 60% usable data points, defined as PHR (Poly High Resolution) and NMH (No Minor Homozygote) marker classes. Thus, an array is needed with a higher percentage of usable data points at a lower cost. We initiated an effort to identify IStraw90 SNP markers that were genetically mapped in one or more strawberry populations from research programs around the world. Seven programs participated in this endeavor. A total of 41,183 SNP probes were submitted to Affymetrix for quality check, 38,506 of which were accommodated on the Axiom® IStraw35 384HT design. In order to assess the performance of the Axiom® IStraw35 384HT array, 384 DNA samples from the University of Florida strawberry breeding program were assayed at a cost of $ 50 per sample, all inclusive. The performance of the array met expectations. More than 87% of markers belonged to the PHR and NMH categories. This array is expected to provide high-quality genome scanning at a more affordable price for strawberry researchers worldwide.

Makino is recognized as an ancestor of the octoploid strawberry species, which includes the cultivated strawberry, × Duchesne ex Rozier. Here we report the construction of the first high-density linkage map for . The linkage map (Fii map) is based on two high-throughput techniques of single nucleotide polymorphism (SNP) genotyping: the IStraw90 Array (hereafter “Array”), and genotyping by sequencing (GBS). The F generation mapping population was derived by selfing hybrid F1D, the product of a cross between two divergent accessions collected from Hokkaido, Japan. The Fii map consists of seven linkage groups (LGs) and has an overall length of 451.7 cM as defined by 496 loci populated by 4173 markers: 3280 from the Array and 893 from GBS. Comparisons with two versions of the ssp. L. ‘Hawaii 4’ pseudo-chromosome (PC) assemblies reveal substantial conservation of synteny and colinearity, yet identified differences that point to possible genomic divergences between and , and/or to genomic assembly errors. The Fii map provides a basis for anchoring a genome assembly as a prerequisite for constructing a second diploid reference genome for .

As the product of interspecific hybridization between its two ancestral octoploid (2n = 8x = 56) species (Fragaria chiloensis and F. virginiana), the cultivated strawberry (F. ×ananassa) is among the most genomically complex of crop plants, harboring subgenomic components derived from as many as four different diploid ancestors. To physically visualize the octoploids’ subgenome composition(s), we launched molecular cytogenetic studies using genomic in situ hybridization (GISH), comparative GISH (cGISH), and rDNA-FISH techniques. First, GISH resolution in Fragaria was tested by using diploid and triploid hybrids with predetermined genome compositions. Then, observation of an octoploid genome was implemented by hybridizing chromosomes of pentaploid (2n = 5x = 35) hybrids from F. vesca × F. virginiana with genomic DNA probes derived from diploids (2n = 2x = 14) F. vesca and F. iinumae, which have been proposed by phylogenetic studies to be closely related to the octoploids yet highly divergent from each other. GISH and cGISH results indicated that octoploid-derived gametes (n = 4x = 28) carried seven chromosomes with hybridization affinities to F. vesca, while the remaining 21 chromosomes displayed varying affinities to F. iinumae, indicating differing degrees of subgenomic contribution to the octoploids by these two putatively ancestral diploids. Combined rDNA-FISH revealed severe 25S rDNA loss in both the F. vesca- and F. iinumae-like chromosome groups, while only the prior group retained its 5S loci.

The cultivated strawberry, Fragaria×ananassa possesses a genetically complex allo-octoploid genome. Advances in genomics research in Fragaria, including the release of a genome sequence for F. vesca, have permitted the development of a high throughput whole genome genotyping array for strawberry, which promises to facilitate genetics and genomics research. In this investigation, we used the Axiom® IStraw90®)array for linkage map development, and produced a linkage map containing 8,407 SNP markers spanning 1,820cM. Whilst the linkage map provides good coverage of the genome of both parental genotypes, the map of 'Monterey' contained significantly fewer mapped markers than did that of 'Darselect'. The array contains a novel marker class known as haploSNPs, which exploit homoeologous sequence variants as probe destabilization sites to effectively reduce marker ploidy. We examined these sites as potential indicators of subgenomic identities by using comparisons to allele states in two ancestral diploids. On this basis, haploSNP loci could be inferred to be derived from F. vesca, F. iinumae, or from an unknown source. When the identity classifications of haploSNPs were considered in conjunction with their respective linkage map positions, it was possible to define two discrete subgenomes, while the remaining homoeologues of each chromosome could not be partitioned into two discrete subgenomic groupings. These findings suggested a novel hypothesis regarding octoploid strawberry subgenome structure and evolutionary origins.

Strawberry (Fragaria spp.) is a valuable fruit crop as well as an outstanding system for studying functional genomics in plants. The goal of this study was to substantially increase and analyze the available expressed sequence information in the genus by examining the transcriptome of the cultivated strawberry (Fragaria × ananassa Duchesne). To maximize transcript diversity and discovery, plants representing an octoploid strawberry cultivar were subjected to a broad range of treatments. Plant materials were pooled by tissue type. cDNA pools were sequenced by the Roche-454 GS-FLX system and assembled into over 32,000 contigs. Predictions of cellular localization and function were made by associating assembled contigs to annotated homologs, and the tissue pool tags provided a means to assess the overall expression pattern for any given transcript. Contigs comprised of reads originating from only one organ type and those present equally in all plant organs were both identifi ed. Bacterial and fungal sequences found in the strawberry samples provide a metagenomic survey of the microbial community of a greenhouse strawberry plant. This study utilized an innovative assembly strategy on pooled tissues, thus providing a foundation for developing tissue-specifi c tools, an opportunity to identify alleles for marker-assisted selection, a reference of strawberry gene annotations, and a basis for comparative transcriptomics between cultivated strawberry, its diploid ancestors, and the wider Rosaceae family. C ULTIVATED STRAWBERRY is a valuable crop, prized for its fl avorful and nutritious fruits. Strawberry is an important crop for many regional economies worldwide and off ers high levels of healthful compounds. Species within the genus Fragaria occur naturally throughout the northern hemisphere and represent many levels of ploidy, including wild diploids and the wild and cultivated octoploids (reviewed in Folta and Davis, 2006; Hummer and Hancock, 2009). Recent attention has been brought to the diploid strawberry species Fragaria vesca Coville because of its utility as a highly tractable experimental system within the economically important Rosaceae family (Folta and Davis, 2006). Strawberry is emerging as a powerful functional genomics system. Fragaria vesca’s haploid genome size of ~200 Mb is among the smallest of the rosaceous crop plants (Shulaev et al., 2008), and its complete genome sequence will be available in 2010, providing an invaluable resource with reference to its octoploid, cultivated relatives. Most importantly, several genotypes of both diploid and octoploid strawberry are readily transformable, making it an outstanding system for functional studies. Protocols exist for diploid and octoploid lines that allow effi cient regeneration of transgenic materials in a period of weeks to months (Folta and Dhingra, 2006; Mezzetti and

Of the three plant genomic components (nuclear, plastid, and mitochondrial) the mitochondrial DNA (mtDNA) has received the least research attention. We describe a phylogenetically informative sequence in the matR gene region of the Fragaria (strawberry) mtDNA, and the first mtDNA marker developed for this economically important genus. The sequence was obtained from a fosmid genomic library clone of F. vesca subsp. americana 'Pawtuckaway'. New mtDNA PCR primer sets work on members of the Rosaceae family, and have potential for broad applicability in the angiosperms. An informative mtDNA CAPS (cleaved amplified polymorphic site) marker provided evidence that mtDNA was transmitted maternally in two interspecific crosses, and that diploid F. iinumae is the likely mtDNA donor to the octoploid species F. chiloensis and F. virginiana, but not to decaploid F. iturupensis. We expanded the database of Fragaria mtDNA sequence by assembling a 67 kb mtDNA contig from Illumina 36 bp paired-end reads from 'Pawtuckaway', providing a basis to develop additional mtDNA markers.

The United States Department of Agriculture (USDA) - Agricultural Research Service (ARS) - National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon, is a genebank that preserves strawberry genetic resources. The Fragaria L. collection consists of more than 1700 accessions of 17 species from 37 countries. In 2004, Asian diploid species, F. iinumae Makino and F. nipponica Makino, were collected during an expedition to Hokkaido, Japan. An ancestor of F. iinumae may be a progenitor of the "B" genome for octoploid strawberry species. The objective of this study was to evaluate the diversity of these two species using microsatellite markers. We report preliminary results obtained from SSR analysis of 139 accessions based on 20 SSRs. A higher genetic diversity was observed in F. nipponica, which tends to be cross pollinated, than in F. iinumae, which tends to be self-pollinated. This study identified putative hybrids between F. iinumae and F. nipponica as well as an unexpected octoploid accession from Hokkaido, Japan. The hybrid nature of these accessions and the possible source of this octoploid accession will be further evaluated using chloroplast and nuclear markers as well as morphological traits.

The genome composition of the octoploid, cultivated strawberry awaits rigorous delineation. Initial insight into the organization and content of strawberry gene space comes from analysis of 50 genomic (fosmid) library inserts totaling ~1.75 Mb from the ~200 Mb genome of the diploid model species Fragaria vesca. Intergenic distances were generally low (0.5 to 5 kb), allowing for amplification of PCR products spanning highly polymorphic intergenic regions. Allele mining at such "gene pair" loci offers a new window onto allelic diversity and genome compositions of octoploids F. ×ananassa, F. chiloensis and F. virginiana. Three distinct haplotype classes were detected at the gRGA1-Subtilase gene pair locus in F. virginiana, providing molecular evidence that its genome is derived from at least three distinct diploid ancestors. Considerable variation was also present within haplotype classes, supporting the expectation that up to eight haplotypes can be differentiated at a single locus in an octoploid individual. A diploid (F. vesca) × octoploid (F. virginiana) cross has yielded a segregating pentaploid population, which is being used to assess patterns of haplotype transmission from the octoploid parent.

The fruit cyanidin (Cyn) and pelargonidin (Plr) contents in 73 Fragaria accessions were assayed using a modified HPLC method, and % Cyn (Cyn as % of Cyn-plus-Plr) values were calculated. Plr content increased during fruit ripening, while Cyn content held steady. Consistent with previous studies, % Cyn was comparatively low (5-20%) among twelve F. ×ananassa cultivars, but was >40% in diploid F. vesca. Importantly, three F. chiloensis accessions had greater than 50% Cyn, and ssp. lucida CFRA1691 had the overall highest Cyn content: 707 μg/g FDW. An F 1 population of 'Bountiful' × 'Pink Panda' segregated for both Cyn and Plr contents. Our investigation identifies useful germplasm, offering promise for increasing Cyn content and antioxidant potential through introgressive breeding.

Physiologically based pharmacokinetic (PBPK) models need the correct organ/tissue weights to match various total body weights in order to be applied to children and the obese individual. Baseline data from Reference Man for the growth of human organs (adrenals, brain, heart, kidneys, liver, lungs, pancreas, spleen, thymus, and thyroid) were augmented with autopsy data to extend the describing polynomials to include the morbidly obese individual (up to 250 kg). Additional literature data similarly extends the growth curves for blood volume, muscle, skin, and adipose tissue. Collectively these polynomials were used to calculate blood/organ/tissue weights for males and females from birth to 250 kg, which can be directly used to help parameterize PBPK models. In contrast to other black/white anthropomorphic measurements, the data demonstrated no observable or statistical difference in weights for any organ/tissue between individuals identified as black or white in the autopsy reports.

Resistance gene analog (RGA) sequences were obtained from four Mentha longifolia (L.) Huds. accessions using degenerate polymerase chain reaction (PCR) primers targeting the conserved nucleotide binding site domain found in many plant disease resistance genes. Seven distinct RGA families were identified. All M. longifolia RGAs showed similarity to sequences of the non-toll-interleukin 1 receptor R gene class. In addition, degenerate PCR primers based on the tomato (Solanum lycopersicum L.) verticillium wilt resistance (Ve) genes were used to PCR-amplify a 445-base pair (bp) Ve-like sequence from M. longifolia that had ≈57% predicted amino acid identity with Ve. Mint-specific primers based on the original mint Ve sequence were used to obtain mint-specific Ve sequences from four M. longifolia accessions and from peppermint (Mentha ×piperita L.) cultivar ‘Black Mitcham’ that had 95% to 100% predicted amino acid identity to the original mint Ve sequence. Inverse PCR was then used to obtain flanking mint Ve sequence from one M. longifolia accession extending the mint Ve sequence to 1077 bp. This is the first report of RGA sequences in the Lamiaceae and the first report of Ve-like sequences obtained with degenerate PCR primers.

This is Scientific Contribution Number 2565 from the New Hampshire Agricultural Experiment Station., [Background]: A high-throughput genotyping platform is needed to enable marker-assisted breeding in the allo-octoploid cultivated strawberry Fragaria × ananassa. Short-read sequences from one diploid and 19 octoploid accessions were aligned to the diploid Fragaria vesca ‘Hawaii 4’ reference genome to identify single nucleotide polymorphisms (SNPs) and indels for incorporation into a 90 K Affymetrix® Axiom® array. We report the development and preliminary evaluation of this array., [Results]: About 36 million sequence variants were identified in a 19 member, octoploid germplasm panel. Strategies and filtering pipelines were developed to identify and incorporate markers of several types: di-allelic SNPs (66.6%), multi-allelic SNPs (1.8%), indels (10.1%), and ploidy-reducing “haploSNPs” (11.7%). The remaining SNPs included those discovered in the diploid progenitor F. iinumae (3.9%), and speculative “codon-based” SNPs (5.9%). In genotyping 306 octoploid accessions, SNPs were assigned to six classes with Affymetrix’s “SNPolisher” R package. The highest quality classes, PolyHigh Resolution (PHR), No Minor Homozygote (NMH), and Off-Target Variant (OTV) comprised 25%, 38%, and 1% of array markers, respectively. These markers were suitable for genetic studies as demonstrated in the full-sib family ‘Holiday’ × ‘Korona’ with the generation of a genetic linkage map consisting of 6,594 PHR SNPs evenly distributed across 28 chromosomes with an average density of approximately one marker per 0.5 cM, thus exceeding our goal of one marker per cM., [Conclusions]: The Affymetrix IStraw90 Axiom array is the first high-throughput genotyping platform for cultivated strawberry and is commercially available to the worldwide scientific community. The array’s high success rate is likely driven by the presence of naturally occurring variation in ploidy level within the nominally octoploid genome, and by effectiveness of the employed array design and ploidy-reducing strategies. This array enables genetic analyses including generation of high-density linkage maps, identification of quantitative trait loci for economically important traits, and genome-wide association studies, thus providing a basis for marker-assisted breeding in this high value crop., This study was partially funded by the USDA’s National Institute of Food and Agriculture – Specialty Crop Research Initiative project, “RosBREED: Enabling marker-assisted breeding in Rosaceae” (2009-51181-05808). Partial funding was also provided by the New Hampshire Agricultural Experiment Station (Projects NH00433 and NH00588 - to TMD), and from USDA-CSREES National Research Initiative (NRI) Plant Genome Grant 2008-35300-04411 (to TMD). Technological Top Institute Green Genetics, project number 1C004RP, is acknowledged for financial support. Financial support was also provided by AGL2010-21414 from the Spanish Ministry of Science (to AM); from the USDA-ARS CRIS number 5358-21000-044-00D (to NVB).

Despite its value as a crop and potential utility as an experimental system, relatively little is known about the molecular-genetic aspects of inheritance or physiology in the cultivated strawberry (Fragaria xananassa). This lack of information exists at a time when biotechnology may offer important remedies to address traditional and contemporary challenges that growers face. An improved understanding of genome structure will hasten the development of molecular markers and unveil clues to the composition of this unique, octoploid genome. Definition of gene function will guide the generation of transgenic resources for research use and possibly toward cultivar development. This review seeks to compile and present the current knowledge state of the molecular-genetic basis of cultivated strawberry genomic form and function. Ongoing studies promise to expand the use of genomic tools and appropriate model systems to rapidly discern the structural and functional basis for traits of interest to agriculture, suc...

The cultivated strawberry, Fragaria ×ananassa Duchesne ex Rozier, originated via hybridization between octoploids F. chiloensis (L.) Mill. and F. virginiana Mill. These three octoploid species are thought to share a putative genome composition of AAA`A'BBB`B'. Diploid F. vesca L., is considered to have donated the A genome. Current attention to the development of a diploid model system for strawberry genomics warrants the assessment of simple sequence repeat (SSR) marker transferability between the octoploid and diploid species in Fragaria L. In the present study, 23 SSR primer pairs derived from F. ×ananassa `Earliglow' by genomic library screening were evaluated for their utility in six diploid Fragaria species, including eight representatives of F. vesca, four of F. viridis Weston, and one each of F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandshurica Staudt, F. iinumae Makino, and F. nilgerrensis Schltdl. ex J. Gay. SSR primer pair functionality, as measured by amplification success rate (= 100% - failure rate) in each species, was ranked (from highest to lowest) as follows: F. vesca (98.4%) > F. iinumae (93.8%) = F. nubicola (93.8%) > F. mandshurica (87.5%) > F. nilgerrensis (75%) > F. viridis (73.4%). The extent to which these octoploid-derived SSR primer pairs generated markers that could be added to the F. vesca linkage map also was assessed. Of the 13 F. ×ananassa SSR markers that segregated codominantly in the F. vesca mapping population, 11 were assigned to linkage groups based upon close linkages to previously mapped loci. These markers were distributed over six of the seven F. vesca linkage groups, and can serve as anchor loci defining these six groups for purposes of comparative mapping between F. vesca and F. ×ananassa.

A set of 41 polymorphic microsatellite markers were developed using a CT/AG-enriched genomic library of Fragaria vesca cv. Reine des Vallées. Thirty-five of them were polymorphic in F. vesca and were tested in one accession each of six additional diploid Fragaria species and the octoploid Fragaria× ananassa. A mean of 5.3 alleles per locus and a low level of observed heterozygosity were generally detected in the 32 single-locus simple sequence repeats of F. vesca. Most of these loci amplify in the other diploid species and in F. × ananassa., Funds for this project were provided by grant no. AGL2003-04691 from CICYT (Spain), and a fellowship to S. Vilanova from MEC of Spain (EX2003-1083).

Mentha longifolia, a wild relative of the polyploid, cultivated Mentha (mint) species, was evaluated as a potential model system for genetic research relevant to the cultivated mints. Fourteen Mentha longifolia accessions maintained by the US Department of Agriculture (USDA), Agricultural Research Service, National Clonal Germplasm Repository (NCGR), were highly diverse with respect to geographic origin, oil composition, verticillium wilt resistance, aspects of morphology, and molecular marker polymorphism. Accession CMEN 584 was the only carvone chemotype, while CMEN 682 was the only accession with high menthol content. Trans-piperitone oxide was the primary oil component of accessions CMEN 17 and CMEN 18, while pulegone was most abundant in CMEN 20, CMEN 500, CMEN 501, and CMEN 585. Four accessions—CMEN 585, CMEN 17, CMEN 501, and CMEN 81—were consistently resistant to verticillium wilt, while CMEN 584 and CMEN 516 were highly susceptible. Pairwise similarity coefficients were calculated and a UPGMA (unweighted pair-group analysis) tree was constructed on the basis of 63 informative randomly amplified polymorphic DNA (RAPD) marker bands. CMEN 585 and CMEN 584 shared the greatest number of bands (16), and formed a distinct cluster in the UPGMA tree. Seven pairs of accessions had no bands in common, emphasizing the high degree of molecular diversity represented by these accessions. The favorable features of diploid (2n = 2x = 24) genome constitution, comparatively small genome size (400 to 500 Mb), self-fertility, fecundity, and diversity with respect to economically relevant traits, contribute to M. longifolia's potential usefulness as a model system for the cultivated mints. As a perennial species amenable to vegetative propagation, M. longifolia's spectrum of susceptibility/resistance to an important vascular wilt disease encourages its further evaluation as a system for broader studies of plant–microbe interactions and disease resistance mechanisms.

Diploid Fragaria provide a potential model for genomic studies in the Rosaceae. To develop a genetic linkage map of diploid Fragaria, we scored 78 markers (68 microsatellites, one sequence-characterised amplified region, six gene-specific markers and three morphological traits) in an interspecific F2 population of 94 plants generated from a cross of F.vesca f. semperflorens x F. nubicola. Co-segregation analysis arranged 76 markers into seven discrete linkage groups covering 448 cM, with linkage group sizes ranging from 100.3 cM to 22.9 cM. Marker coverage was generally good; however some clustering of markers was observed on six of the seven linkage groups. Segregation distortion was observed at a high proportion of loci (54%), which could reflect the interspecific nature of the progeny and, in some cases, the self-incompatibility of F. nubicola. Such distortion may also account for some of the marker clustering observed in the map. One of the morphological markers, pale-green leaf (pg) has not previously been mapped in Fragaria and was located to the mid-point of linkage group VI. The transferable nature of the markers used in this study means that the map will be ideal for use as a framework for additional marker incorporation aimed at enhancing and resolving map coverage of the diploid Fragaria genome. The map also provides a sound basis for linkage map transfer to the cultivated octoploid strawberry.

PURPOSE To determine the efficacy and durability of percutaneous vertebroplasty for the treatment of back pain associated with vertebral body compression fractures. MATERIALS AND METHODS One hundred patients (79 women, 21 men; mean age, 73.7 y) underwent 156 percutaneous injections of polymethylmethacrylate (PMMA) into a vertebra (68 thoracic and 88 lumbar) under fluoroscopic guidance over a 35-month period. Before the procedure and at follow-up, patients were asked to quantify their pain on a visual analog scale (VAS) and complete a follow-up questionnaire of our own design. RESULTS The procedure was technically successful in all patients. There were two complications. One patient sustained a sternal fracture and one experienced a transient radiculopathy. Ninety-seven patients (97%) reported significant pain relief 24 hours after treatment. Mean follow-up duration was 21.5 months (6–44 mo) in 99 patients. Ninety-two patients (93%) reported significant improvement in back pain previously associated with their compression fractures as well as improved ambulatory ability. Before vertebroplasty, the VAS score for the 99 patients was 8.91 ± 1.12 compared to a score of 2.02 ± 1.95 at follow-up. The mean difference in VAS scores was significant ( P CONCLUSION Percutaneous vertebroplasty of symptomatic vertebral body compression fractures is a minimally invasive procedure that provides immediate and sustained pain relief in patients with refractory pain.

The genus Fragaria encompasses species at ploidy levels ranging from diploid to decaploid. The cultivated strawberry, Fragaria×ananassa, and its two immediate progenitors, F. chiloensis and F. virginiana, are octoploids. To elucidate the ancestries of these octoploid species, we performed a phylogenetic analysis using intron-containing sequences of the nuclear ADH-1 gene from 39 germplasm accessions representing nineteen Fragaria species and one outgroup species, Dasiphora fruticosa. All trees from Maximum Parsimony and Maximum Likelihood analyses showed two major clades, Clade A and Clade B. Each of the sampled octoploids contributed alleles to both major clades. All octoploid-derived alleles in Clade A clustered with alleles of diploid F. vesca, with the exception of one octoploid allele that clustered with the alleles of diploid F. mandshurica. All octoploid-derived alleles in clade B clustered with the alleles of only one diploid species, F. iinumae. When gaps encoded as binary characters were included in the Maximum Parsimony analysis, tree resolution was improved with the addition of six nodes, and the bootstrap support was generally higher, rising above the 50% threshold for an additional nine branches. These results, coupled with the congruence of the sequence data and the coded gap data, validate and encourage the employment of sequence sets containing gaps for phylogenetic analysis. Our phylogenetic conclusions, based upon sequence data from the ADH-1 gene located on F. vesca linkage group II, complement and generally agree with those obtained from analyses of protein-encoding genes GBSSI-2 and DHAR located on F. vesca linkage groups V and VII, respectively, but differ from a previous study that utilized rDNA sequences and did not detect the ancestral role of F. iinumae.

A candidate gene approach was used to determine the likely molecular identity of the c locus (yellow fruit color) in Fragaria vesca, a diploid (2n=2x=14) strawberry. Using PCR with degenerate primer pairs, intron-containing segments of structural genes coding for chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and one Del-like regulatory gene in the anthocyanin biosynthetic pathway, were amplified, cloned and sequenced. Intron length polymorphisms for each of these genes were detected among three diploid varieties: F. vesca Alpine variety ’Yellow Wonder’ (YW) (Europe); DN1C, a F. vesca clone collected from Northern California; and Fragaria nubicola FRA520, a U.S.D.A. accession collected in Pakistan. Using F2 generations of the crosses DN1C×YW and YW×FRA520 as mapping populations, the six candidate genes were mapped in relation to previously mapped randomly amplified polymorphic DNA (RAPD) markers and morphological markers. The F3H gene was linked without recombination to the c locus in linkage group I, while the other five candidate genes mapped to different linkage groups. These results suggest that the wild-type allele (C) of the c (yellow fruit color) locus encodes an F3H necessary for red fruit color in F. vesca.

S1 analysis of long PCR heteroduplexes was found to be an effective method for detecting phylogenetically informative indel (insertion/deletion) polymorphisms in the highly conserved strawberry chloroplast genome. In this broadly applicable method, long-range PCR products containing heteroduplex DNA molecules generated from mixed-template amplifications are subjected to S1 nuclease digestion followed by separation and visualization on an agarose gel. The presence of fragments resulting from S1 digestion of mismatch loops in heteroduplex molecules is indicative of indel polymorphism between the template sources. Upon analysis of 13-kb heteroduplex-containing PCR products spanning the petA-psbB chloroplast genome region in Fragaria vesca and Fragaria moschata, two indels distinguishing these species were detected, and subsequently localized to the psbJ-psbL and rpl20-rps18 intergenic regions. Comparative sequencing of these regions revealed that F. moschata resembled Fragaria viridis, but differed from F. vesca, Fragaria nubicola, and a closely related outgroup representative, Duchesnea indica, by a 10-bp deletion in the psbJ-psbL region and a 10-bp insertion in the rpl20-rps18 region. Thus, of the three diploids (2n = 2x = 14) examined, F. viridis is favored over F. vesca and F. nubicola as the most likely chloroplast genome donor to hexaploid (2n = 6x = 42) F. moschata.

Agrobacterium-mediated transformation was used to stably introduce β-glucuronidase (gus) and neomycin phosphotransferase (nptII) marker genes into `Alpine' Fragaria vesca FRA 197, a diploid (2n = 2x = 14) strawberry. R0 generation transformants derived from a single clump of kanamycin-resistant callus were vegetatively propagated. The presence of the gus and nptII genes in five clonal R0 runner plants was confirmed by PCR. Southern analysis suggested two sites of nptII insertion. When R1 generation seedlings obtained via self-fertilization of R0 plants were tested by histochemical assay, 591 were GUS positive and 39 were GUS negative. The R1 segregation data fit a 15 : 1 ratio (0.5 > P > 0.25), consistent with the independent segregation of two transgene insertion loci. These results demonstrate the suitability of `Alpine' F. vesca for transgene research in strawberry.

A 445 cM long genetic linkage map consisting of seven linkage groups was constructed for the diploid (2n = 2x = 14) strawberry, Fragaria vesca. Segregation data used for linkage analysis were obtained from the F2 generation of a cross between Baron Solemacher (BS), an Alpine F. vesca variety, and WC6, an F. vesca clone collected from the wild in New Hampshire. Segregation ratios were systematically skewed in five linkage groups, in all cases favoring the BS alleles over the WC6 alleles. The marker map includes 64 dominant and 11 codominant randomly amplified polymorphic DNA (RAPD) markers, an alcohol dehydrogenase locus detected as a PCR-based sequence tagged site, the phosphoglucose isomerase and shikimate dehydrogenase isozyme loci, and the runnering and fruit color loci. A notable feature of the map is the unusually large number of codominant RAPD markers, the detection of which was due in part to the use of template mixing methods for primer testing and marker analysis. Alternate alleles of a maternally inherited RAPD marker were also detected using these methods.

Ten codominant RAPD markers, ranging in size from about 300 to about 1350 bp, were identified in mapping populations of chickpea (Cicer arietinum L.) and diploid strawberry (Fragaria vesca L.). A distinguishing feature of all ten markers, and perhaps of codominant RAPD markers in general, was the presence in heterozygous individuals of a non-parental, heteroduplex band migrating more slowly than either of the respective parental bands. This non-parental band could also be generated by mixing parental DNAs before PCR (template mixing). As a means of identifying primers likely to detect codominant RAPD markers, parental and mixed-template (parent-parent) PCR-product gel lanes were compared for 20 previously untested RAPD primers (10-base oligomers). Four primers that produced a total of five non-parental, heteroduplex bands in mixed-template reactions were selected, and then used to detect a total of five segregating, codominant markers and nine dominant markers in the respective F2 mapping population, a codominant marker frequency of 35.7%. When closely migrating fast and slow bands of codominant RAPDs were difficult to differentiate, parent-progeny template mixing was used to deliberately generate heteroduplex bands in fast- or slow-band F2 homozygotes, respectively, allowing confirmation of marker phenotype.