Discovery of three loci increasing resistance to charcoal rot caused by Macrophomina phaseolina in octoploid strawberry.

Charcoal rot caused by Macrophomina phaseolinais an increasing economic problem in annualized strawberry production systems around the world. Currently there are no effective postfumigation chemical controls for managing charcoal rot, and no information is available on the genetic architecture of resistance to M. phaseolina in strawberry (Fragaria ×ananassa). In this study, three multiparental discovery populations and two validation populations were inoculated at planting and evaluated for mortality in three consecutive growing seasons. Genome-wide SNP genotyping and pedigree-based analysis with FlexQTL™ software were performed. Two large-effect quantitative trait loci (QTL) increasing charcoal rot resistance were discovered and validated in cultivated germplasm. FaRMp1 was located on linkage group 2A in the interval 20.4to 24.9 cM, while FaRMp2 was located on linkage group 4B in the interval 41.1to 61.2 cM. Together these QTLs explained 27% and 17% of the phenotypic variance in two discovery populations consisting of elite breeding germplasm. For both QTLs, the resistant allele showed some evidence of partial dominance, but no significant interaction was detected between the two loci. As the dosage of resistant alleles increased from 0 to 4 across the two QTLs, mortality decreased regardless of the combination of alleles.A third locus, FaRMp3 on 4D, was discovered in FVC 11-58, a reconstituted F.×ananassa originating from diverse F. virginiana and F. chiloensis accessions. This locus accounted for 44% of phenotypic variation in four segregating crosses. These findings will form the basis for DNA-informed breeding for resistance to charcoal rot in cultivated strawberry.

Long-distance dispersal of the beach strawberry, Fragaria chiloensis, from North America to Chile and Hawaii.

BACKGROUND AND AIMS: The beach strawberry, Fragaria chiloensis, is found in a narrow coastal band from the Aleutian Islands to central California and then jumps thousands of kilometres all the way to Hawaii and Chile. As it probably had a North American origin, it must have been introduced to the other locations by long-distance dispersal. The aim of this study was to determine which agent carried the beach strawberry to its Pacific and South American locations. METHODS: A deductive framework was constructed to separate between the possible modes of long-distance dispersal involving animals, wind and ocean currents. Bird migration was subsequently identified as the most likely scenario, and then the routes, habitats, feeding preferences and flight distances of all the shorebird species were evaluated to determine the most likely carrier. KEY RESULTS: Six species migrate between North America and Chile and feed on the beaches and rocky shores where F. chiloensis grows naturally: Black-bellied Plovers, Greater Yellowlegs, Ruddy Turnstones, Sanderlings, Whimbrels and Willets. Of these, only two eat fruit and migrate in long continuous flight: Ruddy Turnstones and Whimbrels. Two species travel between North America and Hawaii, eat fruit and forage on the beaches and rocky shores where F. chiloensis grows naturally: Pacific Golden-plovers and Ruddy Turnstones. Ruddy Turnstones eat far less fruit than Pacific Golden-plovers and Whimbrels, making them less likely to have introduced the beach strawberry to either location. CONCLUSIONS: We provide evidence that F. chiloesis seeds were probably dispersed to Hawaii by Pacific Golden-plovers and to Chile by Whimbrels.

Influence of intergenotypic competition on multigenerational persistence of abiotic stress resistance transgenes in populations of Arabidopsis thaliana.

Reducing crop losses due to abiotic stresses is a major target of agricultural biotechnology that will increase with climate change and global population growth. Concerns, however, have been raised about potential ecological impacts if transgenes become established in wild populations and cause increased competitiveness of weedy or invasive species. Potential risks will be a function of transgene movement, population sizes, and fitness effects on the recipient population. While key components influencing gene flow have been extensively investigated, there have been few studies on factors subsequent to transgene movement that can influence persistence and competitiveness. Here, we performed multiyear, multigenerational, assessment to examine fitness effects and persistence of three mechanistically different abiotic stress tolerance genes: C-repeat binding factor 3/drought responsive element binding factor 1a (CBF3/DREB1a); Salt overly sensitive 1 (SOS1); and Mannose-6-phosphate reductase (M6PR). Transgenic Arabidopsis thaliana overexpressing these genes were grown in pure populations and in competition with wild-type (WT) parents for six generations spanning a range of field environment conditions. Growth, development, biomass, seed production, and transgene frequency were measured at each generation. Seed planted for each generation was obtained from the previous generation as would occur during establishment of a new genotype in the environment. The three transgenes exhibited different fitness effects and followed different establishment trajectories. In comparison with pure populations, CBF3 lines exhibited reduced dry weight, seed yield, and viable seed yield, relative to WT background. In contrast, overexpression of SOS1 and M6PR did not significantly impact productivity measures in pure populations. In competition with WT, negative fitness effects were magnified. Transgene frequencies were significantly reduced for CBF3 and SOS1 while frequencies of M6PR appeared to be subject to genetic drift. These studies demonstrate the importance of fitness effects and intergenotype competition in influencing persistence of transgenes conferring complex traits.

Clarifying sub-genomic positions of QTLs for flowering habit and fruit quality in U.S. strawberry (Fragaria×ananassa) breeding populations using pedigree-based QTL analysis.

The cultivated strawberry (Fragaria×ananassa) is consumed worldwide for its flavor and nutritional benefits. Genetic analysis of commercially important traits in strawberry are important for the development of breeding methods and tools for this species. Although several quantitative trait loci (QTL) have been previously detected for fruit quality and flowering traits using low-density genetic maps, clarity on the sub-genomic locations of these QTLs was missing. Recent discoveries in allo-octoploid strawberry genomics led to the development of the IStraw90 single-nucleotide polymorphism (SNP) array, enabling high-density genetic maps and finer resolution QTL analysis. In this study, breeder-specified traits were evaluated in the Eastern (Michigan) and Western (Oregon) United States for a common set of breeding populations during 2 years. Several QTLs were validated for soluble solids content (SSC), fruit weight (FWT), pH and titratable acidity (TA) using a pedigree-based QTL analysis approach. For fruit quality, a QTL for SSC on linkage group (LG) 6A, a QTL for FWT on LG 2BII, a QTL for pH on LG 4CII and two QTLs for TA on LGs 2A and 5B were detected. In addition, a large-effect QTL for flowering was detected at the distal end of LG 4A, coinciding with the FaPFRU locus. Marker haplotype analysis in the FaPFRU region indicated that the homozygous recessive genotype was highly predictive of seasonal flowering. SNP probes in the FaPFRU region may help facilitate marker-assisted selection for this trait.

A High-Density Linkage Map of the Ancestral Diploid Strawberry, , Constructed with Single Nucleotide Polymorphism Markers from the IStraw90 Array and Genotyping by Sequencing.

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 .

Public Availability of a Genotyped Segregating Population May Foster Marker Assisted Breeding (MAB) and Quantitative Trait Loci (QTL) Discovery: An Example Using Strawberry.

Much of the cost associated with marker discovery for marker assisted breeding (MAB) can be eliminated if a diverse, segregating population is generated, genotyped, and made available to the global breeding community. Herein, we present an example of a hybrid, wild-derived family of the octoploid strawberry that can be used by other breeding programs to economically find and tag useful genes for MAB. A pseudo test cross population between two wild species of Fragaria virginiana and F. chiloensis (FVC 11) was generated and evaluated for a set of phenotypic traits. A total of 106 individuals in the FVC 11 were genotyped for 29,251 single nucleotide polymorphisms (SNPs) utilizing a commercially available, genome-wide scanning platform (Affymetrix Axiom IStraw90(TW)). The marker trait associations were deduced using TASSEL software. The FVC 11 population segregating for daughters per mother, inflorescence number, inflorescence height, crown production, flower number, fruit size, yield, internal color, soluble solids, fruit firmness, and plant vigor. Coefficients of variations ranged from 10% for fruit firmness to 68% for daughters per mother, indicating an underlying quantitative inheritance for each trait. A total of 2,474 SNPs were found to be polymorphic in FVC 11 and strong marker trait associations were observed for vigor, daughters per mother, yield and fruit weight. These data indicate that FVC 11 can be used as a reference population for quantitative trait loci detection and subsequent MAB across different breeding programs and geographical locations.

Relevance of Crop Biology for Environmental Risk Assessment of Genetically Modified Crops in Africa.

Knowledge about the crop biology of economic crops in Africa is needed for regulators to accurately review dossiers and conduct comprehensive environmental risk assessments (ERAs). This information allows regulators to decide whether biotech crops present a risk to biodiversity, since crossing between domesticated crops and their wild relatives could affect the adaptations of the wild species. The criteria that should be used in the evaluation of African crops for ERA include growth habit, center of origin, center of genetic diversity, proximity of wild relatives, inter-fertility, mode of pollen dispersal, length of pollen viability, mating system, invasiveness, weediness, mode of propagation, mode of seed dispersal, and length of seed dormancy. In this paper, we discuss the crops being genetic engineered in Africa and describe the crop biology of those with native relatives.

Breeding blueberries for a changing global environment: a review.

Today, blueberries are recognized worldwide as one of the foremost health foods, becoming one of the crops with the highest productive and commercial projections. Over the last 100 years, the geographical area where highbush blueberries are grown has extended dramatically into hotter and drier environments. The expansion of highbush blueberry growing into warmer regions will be challenged in the future by increases in average global temperature and extreme fluctuations in temperature and rainfall patterns. Considerable genetic variability exists within the blueberry gene pool that breeders can use to meet these challenges, but traditional selection techniques can be slow and inefficient and the precise adaptations of genotypes often remain hidden. Marker assisted breeding (MAB) and phenomics could aid greatly in identifying those individuals carrying adventitious traits, increasing selection efficiency and shortening the rate of cultivar release. While phenomics have begun to be used in the breeding of grain crops in the last 10 years, their use in fruit breeding programs it is almost non-existent.

The Vaccinium corymbosum FLOWERING LOCUS T-like gene (VcFT): a flowering activator reverses photoperiodic and chilling requirements in blueberry.

KEY MESSAGE: The blueberry FLOWERING LOCUS T ( FT )-like gene ( VcFT ) cloned from the cDNA of a tetraploid, northern highbush blueberry ( Vaccinium corymbosum L.) is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering. ABSTRACT: Blueberry is a woody perennial bush with a longer juvenile period than annual crops, requiring vernalization to flower normally. Few studies have been reported on the molecular mechanism of flowering in blueberry or other woody plants. Because FLOWERING LOCUS T (FT) from Arabidopsis thaliana plays a multifaceted role in generating mobile molecular signals to regulate plant flowering time, isolation and functional analysis of the blueberry (Vaccinium corymbosum L.) FT-like gene (VcFT) will facilitate the elucidation of molecular mechanisms of flowering in woody plants. Based on EST sequences, a 525-bpVcFT was identified and cloned from the cDNA of a tetraploid, northern highbush blueberry cultivar, Bluecrop. Ectopic expression of 35S:VcFT in tobacco induced flowering an average of 28 days earlier than wild-type plants. Expression of the 35S:VcFT in the blueberry cultivar Aurora resulted in an extremely early flowering phenotype, which flowered not only during in vitro culture, a growth stage when nontransgenic shoots had not yet flowered, but also in 6-10-week old, soil-grown transgenic plants, in contrast to the fact that at least 1 year and 800 chilling hours are required for the appearance of the first flower of both nontransgenic 'Aurora' and transgenic controls with the gusA. These results demonstrate that the VcFT is a functional floral activator and overexpression of the VcFT is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.

A novel mannose-based selection system for plant transformation using celery mannose-6-phosphate reductase gene.

To investigate its potential application as a selectable marker for plant transformation, the mannitol producing, celery mannose-6-phosphate reductase gene (M6PR) was transformed into Arabidopsis and tobacco using Agrobacterium tumefaciens-mediated transformation. Mannose-tolerance assays in transgenic materials revealed that the M6PR can act as a selectable marker gene in either a positive or a negative selection mode depending on the plant species. For mannose sensitive species, such as Arabidopsis, expression of M6PR enhanced mannose tolerance and provided a positive selection for transgenic seeds. On medium containing 2 g/L mannose, transgenic seeds germinated, whereas wild type (WT) seeds did not. For mannose-tolerant species, expression of M6PR increased mannose sensitivity in tobacco and enabled a negative selection for transgenic leaves and seeds. Mannose at 30 g/L blanched leaf explants from all 29 transgenic tobacco events with M6PR. In contrast, 30 g/L mannose did not inhibit shoot regeneration from leaf explants of WT or transgenic plants with either an antisense M6PR or a plasmid control. Similarly, mannose at 30 g/L inhibited seed germination of transgenic tobacco seeds with M6PR but not that of WT or transgenic tobacco with either the antisense M6PR or the plasmid control. Northern blot confirmed transcripts of the M6PR in transgenic tobacco, and accumulation of mannitol verified activity of the M6PR in tobacco leaves. Either positive or negative selection using the celery M6PR is versatile for plant transformation. Additionally, the celery M6PR is a potential target gene for improving salt-tolerance in plants due to mannitol accumulation.

Contributions of domesticated plant studies to our understanding of plant evolution.

BACKGROUND: Plant evolutionary theory has been greatly enriched by studies on crop species. Over the last century, important information has been generated on many aspects of population biology, speciation and polyploid genetics. SCOPE: Searches for quantitative trait loci (QTL) in crop species have uncovered numerous blocks of genes that have dramatic effects on adaptation, particularly during the domestication process. Many of these QTL have epistatic and pleiotropic effects making rapid evolutionary change possible. Most of the pioneering work on the molecular basis of self-incompatibility has been conducted on crop species, along with the sequencing of the phytopathogenic resistance genes (R genes) responsible for the 'gene-to-gene' relations of coevolution observed in host-pathogen relationships. Some of the better examples of co-adaptation and early acting inbreeding depression have also been elucidated in crops. Crop-wild progenitor interactions have provided rich opportunities to study the evolution of novel adaptations subsequent to hybridization. Most crop/wild F1 hybrids have reduced fitness, but in some instances the crop relatives have acquired genes that make them more efficient weeds through crop mimicry. Studies on autopolyploid alfalfa and potato have uncovered the means by which polyploid gametes are formed and have led to hypotheses about how multiallelic interactions are associated with fitness and self-fertility. Research on the cole crops and wheat has discovered that newly formed polyploids can undergo dramatic genome rearrangements that could lead to rapid evolutionary change. CONCLUSIONS: Many more important evolutionary discoveries are on the horizon, now that the whole genome sequence is available of the two major subspecies of rice Oryza sativa ssp. japonica and O. sativa ssp. indica. The rice sequence data can be used to study the origin of genes and gene families, track rates of sequence divergence over time, and provide hints about how genes evolve and generate products with novel biological properties. The rice sequence data has already been mined to show that transposable elements often carry fragments of cellular genes. This type of genome shuffling could play a role in creating novel, reorganized genes with new adaptive properties.

Evidence of hybridization between Lythrum salicaria (purple loosestrife) and L. alatum (winged loosestrife) in North America.

BACKGROUND AND AIMS: Although Lythrum salicaria (purple loosestrife) was introduced to North America from Europe in the early 1800s, it did not become invasive until the 1930s. Whether hybridization with L. alatum (winged loosestrife) could have played a role in its ultimate spread was tested. METHODS: Six diagnostic morphological traits (flower number per axil, leaf placement, calyx pubescence, style type, plant height and leaf shape) were surveyed in 30 populations of Lythrum across eastern North America. Patterns of AFLP variation were also evaluated using five primer pairs in a 'global screen' of the same North American populations of L. salicaria and L. alatum described above, in L. salicaria from 11 European populations located in Germany, England, Ireland, Austria and Finland, and in six L. salicaria cultivars. KEY RESULTS: All of the North American L. salicaria populations had individuals with alternate leaf placement and 1-2 flowers per leaf axil, which have not been described in Eurasian L. salicaria but predominate in North American L. alatum. In addition, two L. salicaria populations were intermediate in height and leaf ratio between the typical L. salicaria and L. alatum populations in their native fields and when grown in a common greenhouse. In screens of variation patterns using 279 AFLPs, only two fragments were found that clearly supported introgression from L. alatum to L. salicaria. CONCLUSIONS: The evidence indicates that L. salicaria may have hybridized with L. alatum, but if so, only a small fraction of L. alatum genes have been retained in the genome of L. salicaria. This is unlikely to have led to a dramatic adaptive shift unless the introgression of a few key genes into L. salicaria stimulated a genomic reorganization. It is more likely that crossing among genotypes of L. salicaria from multiple introductions provided the necessary variability for new adaptations to arise.

Commercial Seed Lots Exhibit Reduced Seed Dormancy in Comparison to Wild Seed Lots of Echinacea purpurea.

Seed germination patterns were studied in E. purpurea (L.) Moench grouped by seed source, one group of seven lots from commercially cultivated populations and a second group of nine lots regenerated from ex situ conserved wild populations. Germination tests were conducted in a growth chamber in light (40 µmol·m(-2)·s(-1)) or darkness at 25 °C for 20 days after soaking the seeds in water for 10 minutes. Except for two seed lots from wild populations, better germination was observed for commercially cultivated populations in light (90% mean among seed lots, ranging from 82% to 95%) and in darkness (88% mean among seed lots, ranging from 82% to 97%) than for wild populations in light (56% mean among seed lots, ranging from 9% to 92%) or in darkness (37% mean among seed lots, ranging from 4% to 78%). No germination difference was measured between treatments in light and darkness in the commercially cultivated populations, but significant differences were noted for treatments among wild populations. These results suggest that repeated cycles of sowing seeds during cultivation without treatments for dormancy release resulted in reduced seed dormancy in E. purpurea.

Changes in fruit antioxidant activity among blueberry cultivars during cold-temperature storage.

Antioxidant activity, total phenolic content, anthocyanin content, and six other fruit characters including titratable acid concentration, soluble solids, firmness, and percentage of bruised berries were determined for nine blueberry (Vaccinium L. sp.) cultivars at harvest and at various postharvest intervals after storage at 5 degrees C. Berries from MSU-58, Brigitta, and Legacy stored successfully for 7 weeks, Bluegold stored for 3-5 weeks, Bluecrop, Elliott, and Nelson stored for 3 weeks, and Jersey and Little Giant stored for fewer than 3 weeks. During the time they retained marketable quality, one cultivar (MSU-58) demonstrated a 29% increase in antioxidant activity. None of the cultivars showed a significant decrease from the harvest antioxidant activity value during storage. Antioxidant activity, total phenolic content, and anthocyanin content were strongly correlated with each other (r = 0.87-0.99, P < 0.01). All three parameters were moderately correlated with soluble solids (r = 0.47, P < or =0.05; r = 0.44, P < or = 0.05; and r = 0.64, P < or = 0.01, respectively), and antioxidant activity and total phenolic content were both moderately correlated with pH (r = 0.53 and 0.49, respectively; P < or = 0.05). However, antioxidant activity, total phenolic content, and anthocyanin content showed no correlation with firmness, percent severely bruised berries, or weight loss. Antioxidant activity and total phenolic content at harvest both correlated with titratable acidity at harvest (r = 0.68, P < or = 0.05 and r = 0.70, P < or = 0.05, respectively) on a cultivar mean basis. Berries from Elliott were also harvested from plants at two levels of bush ripeness (30-50% and 60-80% ripe berries on plants) and separated into three fruit maturity classes on the basis of percent blue color. The level of bush ripeness had no significant effect on antioxidant activity, total phenolic content, or anthocyanin content; however, fruit maturity had a significant effect on antioxidant activity, total phenolic content, and anthocyanin content, and bush ripeness x fruit maturity interactions were significant for these three traits. Berries with 50-75% blue coloration harvested from bushes with 60-80% mature fruit showed a significant increase in antioxidant activity, total phenolic content, and anthocyanin content during the first 3 weeks in storage. Our results demonstrate that increases in antioxidant activity, total phenolic content, and anthocyanin content may occur in the blueberry during cold storage and are cultivar-dependent. The increases that occur in immature fruit, such as in Elliott, may be advantageous for producers who wish to delay marketing of the fruit.