Development and bin mapping of a Rosaceae Conserved Ortholog Set (COS) of markers
© Cabrera et al; licensee BioMed Central Ltd. 2009
Received: 19 August 2009
Accepted: 29 November 2009
Published: 29 November 2009
Detailed comparative genome analyses within the economically important Rosaceae family have not been conducted. This is largely due to the lack of conserved gene-based molecular markers that are transferable among the important crop genera within the family [e.g. Malus (apple), Fragaria (strawberry), and Prunus (peach, cherry, apricot and almond)]. The lack of molecular markers and comparative whole genome sequence analysis for this family severely hampers crop improvement efforts as well as QTL confirmation and validation studies.
We identified a set of 3,818 rosaceaous unigenes comprised of two or more ESTs that correspond to single copy Arabidopsis genes. From this Rosaceae Conserved Orthologous Set (RosCOS), 1039 were selected from which 857 were used for the development of intron-flanking primers and allele amplification. This led to successful amplification and subsequent mapping of 613 RosCOS onto the Prunus TxE reference map resulting in a genome-wide coverage of 0.67 to 1.06 gene-based markers per cM per linkage group. Furthermore, the RosCOS primers showed amplification success rates from 23 to 100% across the family indicating that a substantial part of the RosCOS primers can be directly employed in other less studied rosaceaous crops. Comparisons of the genetic map positions of the RosCOS with the physical locations of the orthologs in the Populus trichocarpa genome identified regions of colinearity between the genomes of Prunus-Rosaceae and Populus-Salicaceae.
Conserved orthologous genes are extremely useful for the analysis of genome evolution among closely and distantly related species. The results presented in this study demonstrate the considerable potential of the mapped Prunus RosCOS for genome-wide marker employment and comparative whole genome studies within the Rosaceae family. Moreover, these markers will also function as useful anchor points for the genome sequencing efforts currently ongoing in this family as well as for comparative QTL analyses.
The Rosaceae is an important plant family that includes more than 90 genera and 3000 species. The family belongs to the Rosid clade and is closely related to the Salicaceae (including poplar), Leguminoseae (including Medicago and soybean), Cucurbitaceae (including cucumber and melon) and more distantly related to the Brassicaceae (including Arabidopsis). The Rosaceae is divided into three subfamilies, two of which include some of the most economically important temperate fruit crops . The largest subfamily is the Spiraoideae to which Malus (apple), Pyrus (pear) and Prunus (peach, cherry, almond, apricot) belong. The second largest subfamily is the Rosoideae to which Fragaria (strawberry), Rubus (currants, blackberries, raspberries) and Rosa (rose) belong. Within the family, apple, peach and strawberry have been utilized as model species for Rosaceae biology, genetics and genomics .
Comparative analyses of plant genomes offer insights into genome evolution and speciation of closely as well as more distantly related species. In particular, knowledge of the extent and locations of syntenic blocks and chromosomal rearrangements enables the transfer of genomic information among species. This information would aid genome-wide as well as targeted marker development for the identification and validation of loci controlling traits that are important for crop improvement. Without the availability of several sequenced plant genomes within one family, comparative analyses often rely on molecular markers that are shared among the species. One of the earliest efforts towards the construction of comparative plant maps using molecular markers was conducted in the Solanaceae family. Assessment of the degree of similarity between tomato and pepper [3, 4] and tomato and potato  show that the more closely related species, tomato and potato, underwent fewer rearrangements compared to the more distantly related tomato and pepper. Similarly in the Poaceae family, conservation of large chromosomal regions between wheat, barley and rye genomes have been identified [6, 7]. The application of comparative sequence analysis within the grasses greatly facilitated the positional cloning of important genes such as VRN1 from wheat, a species for which map-based cloning was deemed impossible due to its large genome size and the presence of many repetitive elements that would hamper chromosome walking efforts .
Despite the lack of extensive investigations, the potential for comparative genome analysis within the Rosaceae family has been demonstrated by several studies. Genome colinearity was found among Prunus species [9–16]. These comparative studies were based on the Prunus reference map (x = 8), the most detailed genetic map in the Rosaceae, that is derived from an interspecific almond (P. dulcis) cv. Texas × peach (P. persica) cv. Earlygold (abbreviation TxE) F2 mapping population . Good colinearity and marker transferability within the family was also demonstrated by the identification of syntenic regions of the Malus and Prunus genomes [9, 17], and between the more distant genera Prunus and Fragaria [18, 19]. However, a comprehensive and extensive comparative map such as those that were constructed in the Solanaceae and Poaceae families has not been achieved for the Rosaceae. This is mostly due to the lack of conserved markers to apply across the entire family [12, 18].
Genes that are highly conserved and are present as low or single copy in genomes are particularly useful as markers for genome evolution studies as well as whole genome comparative analyses [20, 17]. A Conserved Ortholog Set (COS) is defined as a collection of genes that are conserved in sequence and copy number throughout plant evolution . In contrast, paralogs represent duplicated regions within the genome as a result of single gene duplications and/or large scale polyploidization events . The development of markers from single copy and conserved genes is critical in comparative mapping studies as these markers enable an unambiguous determination of the degree of synteny . In addition, the single copy conserved genes reduce the possibility of erroneously identifying chromosomal rearrangements that could result from mapping paralogous genes .
Complete whole-genome sequence information of model plants together with improved genomic resources from other species, such as EST databases, provide the opportunity for the in silico identification of candidate COS. Using the Arabidopsis whole genome sequence and the EST databases of potato, tomato and pepper, Wu et al identified 2869 Solanaceaous COS . Likewise, a universal set of COS markers was developed for the Asteraceae family after comparing EST from sunflower and lettuce against the whole genome of Arabidopsis . Moreover, comparative genome sequence analysis between the three sequenced model species, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa resulted in the identification of 753 COS candidates among the angiosperms of which 55 to 359 could be identified from pairwise comparisons among four gymnosperm EST databases . Once developed, COS markers have been widely employed to link the genomes of related species within families [20, 26–29]
In this study, we report the first step towards a comprehensive and dense comparative genetic map for rosaceous species. We present the development of a set of conserved Rosaceae gene-based sequences corresponding to single copy Arabidopsis genes. These Rosaceae COS (RosCOS) were subsequently mapped using the bin map population corresponding to the Prunus TxE reference map [10, 30]. Our analyses show that nearly all of the mapped RosCOS are present once in the Prunus genome suggesting that this genus did not undergo a hitherto unknown recent polyploidization event. Additionally, we compared the genetic location of these RosCOS to the physical location of the poplar and Arabidopsis orthologs. These analyses identified many regions that exhibited synteny between Prunus and poplar and to a lesser extent to Arabidopsis.
Results and Discussion
Construction of the RosCOS set
Number of Rosaceae ESTs from different subfamilies and genera.
EST corresponding to Arabidopsis COS
Due to single pass sequencing of EST clones, the chance of sequencing errors can be considerable. In an effort to avoid the design of primers in regions of poor sequence quality, we focused on the 3,818 unigenes that were represented by at least two ESTs. Moreover, contigs tended to have more sequence information (i.e. longer sequences) which was helpful in the design of primers flanking the predicted intron sites. Each contig was named RosCOS### to indicate that this was the set of putatively conserved orthologous Rosaceae sequences. We narrowed the collection down further by selecting RosCOS that were represented by at least two of the three key genera in the family or Prunus alone (see Additional file 1). This selection was chosen to enhance the chance of successful amplification of Prunus DNA with the designed primers because of our goal to map these RosCOS on the Prunus reference map. The reduction led to the final data set of 1,039 RosCOS (Figure 1). We noticed that contigs harboring ESTs from more than one genus usually exhibited a higher number of mismatches in Fragaria than in Malus or Prunus which is consistent with the greater phylogenetic distance between Fragaria and the other two genera .
Amplification and mapping of RosCOS in Prunus
Amplification and bin mapping success for 857 RosCOS primer pairs.
Amplification in TxE1
Putative bin 4:183
RosCOS marker density on the eight Prunus TxE linkage groups.
cM length of the linkage group
Number of RosCOS mapped
RosCOS density per cM
Number of Arabidopsis single copy genes corresponding to more than one RosCOS and their Prunus bin map co-localization.
Number of Arabidopsis genes that correspond more than one RosCOS
RosCOS bin map locations
Map to the same bin
Map to separate bins1
Synteny between Rosaceae, Arabidopsis and Populus
Amplification of RosCOS across the Rosaceae
Amplification success of RosCOS primers in different genera.
Genera represented in each RosCOS
RosCOS per Group
Amplification in Malus
Amplification in Fragaria
Amplification in Prunus(cherry)
Fragaria, Malus and Prunus
Fragaria and Prunus
Fragaria and Malus
Prunus and Malus
Comparative genome analysis for the Rosaceae family lags behind that of other economically important families such as the Solanaceae and Poaceae. The RosCOS resource developed in this study aims to ameliorate this situation by providing a marker set that can be employed for comparative mapping and marker development as well as whole genome comparative analyses in the Rosaceae family. The extensive colinearity observed between poplar and Prunus demonstrates the possibility of additional marker development in targeted regions of the Prunus genome based on synteny with poplar. Moreover, with the advent of Rosaceae species whole genome sequence information that will become available in the near future, these RosCOS will be instrumental to place unlinked scaffolds onto genetic maps and enable marker development to targeted regions in species whose genome is not sequenced. Excellent genetic maps and whole genome sequence data are extremely important for QTL discovery and validation. Therefore, the RosCOS resource developed herein has great potential to benefit rosaceaous crop improvement.
Identification of the Rosaceae COS (RosCOS) set
The set of 3,790 Arabidopsis single copy genes was selected as previously described . The complete data set of 412,827 Rosaceae ESTs as of December 2007 was downloaded from NCBI GenBank  and compared to the Arabidopsis single copy gene set using the BLASTX function at the cutoff E-value of 1e-15. The resulting Rosaceae ESTs were assembled using the Contig Assembly Program: CAP3  with parameters of at least 80 bp overlap and 90% sequence identity. This resulted in the assembly of 7,247 unigenes (3,818 contigs and 3,429 singletons) (Figure 1). The 3,818 contigs were assigned a RosCOS number whereas the singletons were not. The consensus sequence for each RosCOS is found under the name ROSC_FMLY_CSA1_1 beginning with RosCOS 1 . The ESTs that are part of the RosCOS are found in the "December 2007 Assembly Info" . The list of single copy Arabidopsis genes and corresponding RosCOS are found under the "December 2007 BLAST info" links . Information about the final list of RosCOS used in this study can be found under the "RosCOS final selection and QC BLAST" links . RosCOS map and primer data is also available from our own database  as well as in Additional file 2. Sequence data of the peach parent 'Earlygold' has been deposited in GSS at Genbank  and the corresponding accession numbers are listed in Additional file 2 (sheet 2).
Design of PCR primers flanking introns
Orthologous genes share conserved structures such that the position of the introns is conserved . To reduce the probability of sequencing errors in the ESTs and to increase the amplification success rate in multiple Rosaceae species, singletons were discarded from the analysis. Rosaceae contigs comprised of ESTs from three (Fragaria, Malus and Prunus) and two (Fragaria-Malus, Prunus-Fragaria, and Malus-Prunus) genera as well as only Prunus ESTs were selected totaling up to 1,039 RosCOS that were further investigated. The RosCOS were aligned to the Arabidopsis genome and putative intron sites were identified using the Python Contig Viewer program  (Figure 3). Based on the RosCOS sequence length and predicted intron position of these 1039 RosCOS, 857 intron-flanking primer pairs were developed using Primer3 v0.4.0 . Subsequently, all forward primers were designed with an additional M13 tail (CACGACGTTGTAAAACGAC) at the 5' end to facilitate high-throughput direct sequencing of the amplicons.
PCR conditions and polymorphism detection of RosCOS
The RosCOS putative intron-flanking primers were used to amplify the peach parent 'Earlygold', F1 and 6 bin set individuals selected from the Prunus TxE F2 reference population [10, 30]. The amplification reactions were conducted in 96-well plate format in 60 ul reaction volume consisting of 10 mM Tris-Cl pH 8.3, 50 mM KCl, 2 mM MgCl2, 10-100 ng of genomic DNA, 0.1 mM of each dNTP, 0.1 uM of each primer, and 0.25 U Taq polymerase. The reactions were preheated at 94°C for 1 min followed by 31 cycles of 92°C (30 s), 56°C (30 s), 72°C (30 s), and a final extension of 72°C (60 s). Amplified fragments were sequenced using the M13F primer at the Agencourt Bioscience Corporation (Agencourt, Beverly, MA, USA). The sequencing results were analyzed for polymorphisms such as single nucleotide polymorphism (SNP) and/or insertion-deletions (InDels) using Sequencher software v4.2 (Gene Codes Corporation). The presence of a double peak in an otherwise high-quality chromatogram was indicative of the presence of a SNP. The sudden decay of high-quality chromatogram was indicative of the presence of an InDel.
Genotyping and Mapping
Bins representing the different regions of the Prunus genome have been identified by the genotype of a subset of plants from the TxE F2 population . RosCOS markers with a segregation pattern corresponding to a bin set score were grouped in that bin. RosCOS that mapped in bin 2:45 or 3:04 and 5:41 or 8:30, respectively, were analyzed in a 7th genotype to map them in one or the other bin. RosCOS markers that clearly segregated but did not fall into a known bin were categorized as "orphan" RosCOS markers.
Synteny of RosCOS and Poplar COS
The translated sequence of Arabidopsis single copy genes corresponding to the bin-mapped RosCOS were compared to the Populus trichocarpa genome. Using the P. trichocarpa v1.1 genome browser , the physical position of each poplar COS was identified through the TBLASTN function with the cut off E-value of 1e-5. Syntenic blocks between Prunus and poplar were established under the condition that a minimum of three linked RosCOS corresponded to poplar COS that were located within 2 Mb from each other.
This work is supported by USDA-NRI grants 2008-02259 and 2005-00743. AC was also supported by funds from the Department of Horticulture and Crop Science, The Ohio State University. The authors would like to thank Drs. Dan Sargent and David Chagne for providing DNAs from strawberry and apple, respectively.
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