Developing expressed sequence tag libraries and the discovery of simple sequence repeat markers for two species of raspberry (\u3cem\u3eRubus\u3c/em\u3e L.)

Background Due to a relatively high level of codominant inheritance and transferability within and among taxonomic groups, simple sequence repeat (SSR) markers are important elements in comparative mapping and delineation of genomic regions associated with traits of economic importance. Expressed sequence tags (ESTs) are a source of SSRs that can be used to develop markers to facilitate plant breeding and for more basic research across genera and higher plant orders. Methods Leaf and meristem tissue from ‘Heritage’ red raspberry (Rubus idaeus) and ‘Bristol’ black raspberry (R. occidentalis) were utilized for RNA extraction. After conversion to cDNA and library construction, ESTs were sequenced, quality verified, assembled and scanned for SSRs. Primers flanking the SSRs were designed and a subset tested for amplification, polymorphism and transferability across species. ESTs containing SSRs were functionally annotated using the GenBank non-redundant (nr) database and further classified using the gene ontology database. Results To accelerate development of EST-SSRs in the genus Rubus (Rosaceae), 1149 and 2358 cDNA sequences were generated from red raspberry and black raspberry, respectively. The cDNA sequences were screened using rigorous filtering criteria which resulted in the identification of 121 and 257 SSR loci for red and black raspberry, respectively. Primers were designed from the surrounding sequences resulting in 131 and 288 primer pairs, respectively, as some sequences contained more than one SSR locus. Sequence analysis revealed that the SSR-containing genes span a diversity of functions and share more sequence identity with strawberry genes than with other Rosaceous species. Conclusion This resource of Rubus-specific, gene-derived markers will facilitate the construction of linkage maps composed of transferable markers for studying and manipulating important traits in this economically important genus

Comparative analysis of rosaceous genomes and the reconstruction of a putative ancestral genome for the family

Abstract Background Comparative genome mapping studies in Rosaceae have been conducted until now by aligning genetic maps within the same genus, or closely related genera and using a limited number of common markers. The growing body of genomics resources and sequence data for both Prunus and Fragaria permits detailed comparisons between these genera and the recently released Malus × domestica genome sequence. Results We generated a comparative analysis using 806 molecular markers that are anchored genetically to the Prunus and/or Fragaria reference maps, and physically to the Malus genome sequence. Markers in common for Malus and Prunus, and Malus and Fragaria, respectively were 784 and 148. The correspondence between marker positions was high and conserved syntenic blocks were identified among the three genera in the Rosaceae. We reconstructed a proposed ancestral genome for the Rosaceae. Conclusions A genome containing nine chromosomes is the most likely candidate for the ancestral Rosaceae progenitor. The number of chromosomal translocations observed between the three genera investigated was low. However, the number of inversions identified among Malus and Prunus was much higher than any reported genome comparisons in plants, suggesting that small inversions have played an important role in the evolution of these two genera or of the Rosaceae.Apple genome research at FEM is supported by the research office of the Provincia autonoma di Trento. DJS and ELG acknowledge a grant from the East Malling Trust. Fragaria genomics at EMR is funded by the BBSRC. JMB is supported by a grant by Plant & Food Research's Excellence Programme. Apple genomics at Plant & Food Research is partially supported by the New Zealand Foundation for Research Science and Technology project C06X0812 "Exploiting Opportunities from Horticultural Genomics". Research conducted at IRTA was partly funded by the CONSOLIDER-INGENIO 2010 Program (CSD2007-00036) and project INIA-RTA2007-00063-00-00, both from the Spanish Ministry of Science and Innovation. RosCOS development at OSU/MSU was funded by the National Research Initiative Competitive Grant 2005-35300-15454 of USDA's National Institute of Food and Agriculture.Peer Reviewe

Comparative genome mapping of the Rosaceae : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Molecular Genetics and Genomics, Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand

Comparative genome mapping uses genetic map and DNA sequence alignment to assess genome conservation between two or more organisms. This study makes use of the recent genome sequence availability of four Rosaceae genera, and the development of new, and the expansion of existing, linkage maps to: 1) explore overall genome synteny between apple and strawberry; 2) assess homology between, and the degree of ancestral genome rearrangement among, four genera; and 3) compare genome synteny with respect to the production of anthocyanins between raspberry and strawberry. The inter-tribal comparison of the genomes of apple and diploid strawberry, conducted by adding 56 newly developed orthologous markers to existing linkage maps, identified 21 regions of genomic synteny between the linkage groups of apple and strawberry. In addition, this work identified two each of potential translocations, inversions and insertions, and provided a set of orthologous markers that will be useful for orienting and anchoring other Rosaceae genome sequences. Orthologous- and other DNA sequence-based markers were used in the construction of new linkage maps for Rubus occidentalis 96395S1 and R. idaeus =Latham‘. The sequences from which the Rubus markers were designed were compared with the draft genome sequences of Malus × domestica =Golden Delicious‘, Fragaria vesca =Hawaii 4‘, and Prunus persica =Lovell‘ to identify regions of orthology. This first comparison of Rubus linkage maps with other members of the Rosaceae identified a nearly 1:1 homology between the linkage groups of Rubus and F. vesca, as well as family-wide conservation among some linkage groups. The F1 progeny of Rubus occidentalis 96395S1 × R. idaeus =Latham‘ was used to conduct a quantitative trait locus (QTL) analysis to explore the presence of associations between genotype and the variation in concentrations of anthocyanins in the fruit. Seven associations of traits with markers designed from the sequences of transcription factors and anthocyanin biosynthetic pathway genes were identified, providing opportunities for further fine-scale mapping, as well as cloning and expression analyses. The comparison of QTL maps of Rubus and Fragaria × ananassa suggests that homologous genomic regions may be important in the expression of various fruit quality traits

Additional file 3: of Developing expressed sequence tag libraries and the discovery of simple sequence repeat markers for two species of raspberry (Rubus L.)

Gene ontology term distribution for the categories Biological Process, Molecular Function, and Cellular Component. (XLSX 12 kb

Additional file 1: of Developing expressed sequence tag libraries and the discovery of simple sequence repeat markers for two species of raspberry (Rubus L.)

NCBI accession, locus name, and details of SSR, primer design and DNA sequence for red raspberry (R. idaeus). Highlight indicates those loci tested in R. idaeus and R. occidentalis genotypes with results shown in manuscript Table 1. (XLSX 48 kb

Additional file 2: of Developing expressed sequence tag libraries and the discovery of simple sequence repeat markers for two species of raspberry (Rubus L.)

NCBI accession, locus name, and details of SSR, primer design and DNA sequence for black raspberry (R. occidentalis). Highlight indicates those loci tested in R. idaeus and R. occidentalis genotypes with results shown in manuscript Table 1. (XLSX 93 kb

Additional file 1: of Developing expressed sequence tag libraries and the discovery of simple sequence repeat markers for two species of raspberry (Rubus L.)

NCBI accession, locus name, and details of SSR, primer design and DNA sequence for red raspberry (R. idaeus). Highlight indicates those loci tested in R. idaeus and R. occidentalis genotypes with results shown in manuscript Table 1. (XLSX 48 kb

Additional file 4: of Developing expressed sequence tag libraries and the discovery of simple sequence repeat markers for two species of raspberry (Rubus L.)

Top ten longest unigenes aligned to the Gene Ontology database with BLAST results. (XLSX 10 kb

Additional file 3: of Developing expressed sequence tag libraries and the discovery of simple sequence repeat markers for two species of raspberry (Rubus L.)

Gene ontology term distribution for the categories Biological Process, Molecular Function, and Cellular Component. (XLSX 12 kb

Additional file 4: of Developing expressed sequence tag libraries and the discovery of simple sequence repeat markers for two species of raspberry (Rubus L.)

Top ten longest unigenes aligned to the Gene Ontology database with BLAST results. (XLSX 10 kb