Thomas M. Davis

The genome of woodland strawberry ( Fragaria vesca )

The woodland strawberry, Fragaria vesca (2n = 2x = 14), is a versatile experimental plant system. This diminutive herbaceous perennial has a small genome (240 Mb), is amenable to genetic transformation and shares substantial sequence identity with the cultivated strawberry (Fragaria × ananassa) and other economically important rosaceous plants. Here we report the draft F. vesca genome, which was sequenced to ×39 coverage using second-generation technology, assembled de novo and then anchored to the genetic linkage map into seven pseudochromosomes. This diploid strawberry sequence lacks the large genome duplications seen in other rosids. Gene prediction modeling identified 34,809 genes, with most being supported by transcriptome mapping. Genes critical to valuable horticultural traits including flavor, nutritional value and flowering time were identified. Macrosyntenic relationships between Fragaria and Prunus predict a hypothetical ancestral Rosaceae genome that had nine chromosomes. New phylogenetic analysis of 154 protein-coding genes suggests that assignment of Populus to Malvidae, rather than Fabidae, is warranted.

Multiple Models for Rosaceae Genomics

The plant family Rosaceae consists of over 100 genera and 3,000 species that include many important fruit, nut, ornamental, and wood crops. Members of this family provide high-value nutritional foods and contribute desirable aesthetic and industrial products. Most rosaceous crops have been enhanced by human intervention through sexual hybridization, asexual propagation, and genetic improvement since ancient times, 4,000 to 5,000 B.C. Modern breeding programs have contributed to the selection and release of numerous cultivars having significant economic impact on the U.S. and world markets. In recent years, the Rosaceae community, both in the United States and internationally, has benefited from newfound organization and collaboration that have hastened progress in developing genetic and genomic resources for representative crops such as apple (Malus spp.), peach (Prunus spp.), and strawberry (Fragaria spp.). These resources, including expressed sequence tags, bacterial artificial chromosome libraries, physical and genetic maps, and molecular markers, combined with genetic transformation protocols and bioinformatics tools, have rendered various rosaceous crops highly amenable to comparative and functional genomics studies. This report serves as a synopsis of the resources and initiatives of the Rosaceae community, recent developments in Rosaceae genomics, and plans to apply newly accumulated knowledge and resources toward breeding and crop improvement.

Strawberry Genes and Genomics

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, such as those associated with disease, ripening, and volatile production. Together these studies bring new molecular tools to dissect complex traits, implement marker-assisted selection and address important physiological questions in the cultivated strawberry, the Fragaria genus, and the Rosaceae family.

Molecular identification of the yellow fruit color (c) locus in diploid strawberry: a candidate gene approach

Abstract  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.

Template mixing: a method of enhancing detection and interpretation of codominant RAPD markers

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.

Identification of RAPD markers linked to a Phytophthora fragariae resistance gene (Rpf1) in the cultivated strawberry

Abstract Bulked segregant analysis (BSA) was used to identify seven random amplified polymorphic DNA (RAPD) markers linked to the Rpf 1 gene. Rpf 1 confers resistance to Phytophthora fragariae var. fragariae, the causal agent of red stele root rot in Fragaria spp. The bulked DNAs represented subsets of a F1 population obtained from the cross Md683×Senga Sengana which consisted of 60 plants and segregated in a 1:1 ratio for resistance or susceptibility to race 2.3.4 isolate NS2 of P.  fragariae. Seven markers were shown to be linked to Rpf 1 and were generated from four primers; five of these markers were in coupling phase and two in repulsion phase with respect to the gene. A linkage map of this resistance gene region was generated using JoinMap 2.0TM. The manner in which Rpf 1 and the linked markers co-segregated indicated that they are inherited in a disomic fashion. These markers could enable gene pyramiding and marker-assisted selection of resistance genes in strawberry breeding programmes.

Zeatin-induced shoot regeneration from immature chickpea (Cicer arietinum L.) cotyledons

For the purpose of developing an in vitro regeneration system for chickpea (Cicer arietinum L.), an important food legume, immature cotyledons approximately 5 mm long were excised from developing embryos and cultured on B5 basal medium supplemented with 1.5% sucrose and various growth regulator combinations. Only non-morphogenic callus was formed in response to concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) previously reported to induce somatic embryogenesis on immature soybean cotyledons. However, 4.6, 13.7, and 45.6 μM zeatin induced formation of white, cotyledon-like structures (CLS) at the proximal end of immature cotyledons placed with adaxial surface facing the agar medium. No morphogenesis, or occasional formation of fused, deformed CLS, was observed when zeatin was replaced with kinetin or 6-benzyladenine, respectively. The highest response frequency, 64% of explants forming CLS, was induced by 13.7 μM zeatin plus 0.2 μM indole-acetic acid (IAA). Within 20–40 days culture on zeatin, shoots formed at the base of CLS on approximately 50% of CLS-bearing explants, and proliferated upon subsequent transfer to basal medium with 4.4 μM BA or 4.6 μM kinetin. This regeneration system may be useful for genetic transformation of chickpea.

Simple Sequence Repeat Marker Development and Mapping Targeted to Previously Unmapped Regions of the Strawberry Genome Sequence

The genome sequence of the woodland strawberry (Fragaria vesca L.) is an important resource providing a reference for comparative genomics studies and future sequenced rosaceous species and has great utility as a model for the development of markers for mapping in the cultivated strawberry Fragaria ×ananassa Duchesne ex Rozier. A set of 152 microsatellite simple sequence repeat (SSR) primer pairs was developed and mapped, along with 42 previously published but unmapped SSRs, permitting the precise assignment of 28.2 Mbp of previously unanchored genome sequence scaffolds (13% of the F. vesca genome sequence). The original ordering of F. vesca sequence scaffolds was performed without a physical map, using predominantly SSR markers to order scaffolds via anchoring to a comprehensive linkage map. This report complements and expands resolution of the Fragaria spp. reference map and refines the scaffold ordering of the F. vesca genome sequence using newly devised tools. The results of this study provide two significant resources: (i) the concurrent validation of a substantial set of SSRs associated with these previously unmapped regions of the Fragaria spp. genome and (ii) the precise placement of previously orphaned genomic sequence. Together, these resources improve the resolution and completeness of the strawberry genome sequence, making it a better resource for downstream studies in Fragaria spp. and the family Rosaceae.

Gene Content and Distribution in the Nuclear Genome of Fragaria vesca

Thirty fosmids were randomly selected from a library of Fragaria vesca subsp. americana (cv. Pawtuckaway) DNA. These fosmid clones were individually sheared, and ∼4‐ to 5‐kb fragments were subcloned. Subclones on a single 384‐well plate were sequenced bidirectionally for each fosmid. Assembly of these data yielded 12 fosmid inserts completely sequenced, 14 inserts as 2 to 3 contiguous sequences (contigs), and 4 inserts with 5 to 9 contigs. In most cases, a single unambiguous contig order and orientation was determined, so no further finishing was required to identify genes and their relative arrangement. One hundred fifty‐eight genes were identified in the ∼1.0 Mb of nuclear genomic DNA that was assembled. Because these fosmids were randomly chosen, this allowed prediction of the genetic content of the entire ∼200 Mb F. vesca genome as about 30,500 protein‐encoding genes, plus >4700 truncated gene fragments. The genes are mostly arranged in gene‐rich regions, to a variable degree intermixed with transposable elements (TEs). The most abundant TEs in F. vesca were found to be long terminal repeat (LTR) retrotransposons, and these comprised about 13% of the DNA analyzed. Over 30 new repeat families were discovered, mostly TEs, and the total TE content of F. vesca is predicted to be at least 16%.

A Transcript Accounting from Diverse Tissues of a Cultivated Strawberry

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 identified. 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‐specific 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.