Full Text - A molecular marker approach using intron flanking EST-PCR to map candidate genes in peach (Prunus persica)

A molecular marker approach using intron flanking EST-PCR to map candidate genes in peach (Prunus persica)

¹Universidad de Chile, Facultad de Ciencias Agronómicas, Departamento de Producción Agrícola, Laboratorio de Genómica Funcional & Bioinformática, Santiago, Chile

Financial support: This research was supported by Consorcio BIOFRUTALES S.A., PBCT R-11, Millennium Nucleus in Plant Cell Biotechnology (PCB) ICM P06-065-F and CONICYT Fellowship D-2108007 to SDM.

In Peach (Prunus persica) several physiological changes, such as woolliness, triggered by chilling injury are involved in major production losses due to cold storage of the fruits during shipping. Additionally, the low level of polymorphisms among peach varieties is an important limitation in the search for new molecular markers that could be associated with economically important traits. Therefore, a functional approach was employed to associate candidate genes with an informative marker in peach. The data was obtained from the results of an in silico analysis of four different cold peach treatments. Thirty two candidate genes were selected that were aligned against Arabidopsis thaliana genomic sequences to design intron-flanking EST-PCR markers. These markers were used to position the candidate genes on the Prunus genetic reference map. In the physiological response to chilling injury, cell wall integrity, carbohydrate metabolism and stress response pathways could be involved, therefore candidate genes associated by Gene Ontology annotation to these pathways were included in the analysis. The designed markers were positioned to the Texas X Earlygold (TxE) genetic reference map through selective mapping methodology (Bin mapping). 72% of these new markers showed polymorphism in the TxE Binset population and 31% of them were successfully mapped to a genetic position on the Prunus reference map. The bioinformatic methodology used in this work includes a first approach in search for functional molecular markers associated to differentially expressed genes under certain physiological condition which in addition to the Bin mapping approach allows addressing a genetically anchored position to these new markers.

Peach (Prunus persica) is one of the most important fruit exported by Chilean industry. Its production was 100,318 tons in 2011 with a 112.3 million dollars market (Bravo, 2012). Since fruit quality is critical to sustain the position of the product in the market, post harvest alterations are key issue. Due to the long term shipment, fruits are under several adverse conditions that include biotic and abiotic stress, causing considerable damages to the final product, such as woolliness (Lill et al. 1989; Lurie and Crisosto, 2005). The main problem associated with exportation of peaches is related with the woolly phenotype caused by the low temperatures in which the fruit must be storage in order to delay ripening. In this frame of time many cellular processes are triggered, therefore a fine regulation is needed (Lill et al. 1989; Lurie and Crisosto, 2005). Approaches to manage these conditions have been reported. Intermittent warming (Fernández-Trujillo and Artés, 1997; Lurie and Crisosto, 2005) and pre conditioning (Infante et al. 2009) has been used. These two approaches involved temperature changes through the fruit cold chain transport, which is directly associated with additional costs.

Alternative approaches to try to solve this problem are Molecular Assisted Selection (MAS) and transgenic plant technologies. In order to find a solution that could be used in a MAS program, identification of genes involved in this physiological problem in peach is needed. Several approaches have been taken, such as candidate genes identification (Horn et al. 2005; González-Agüero et al. 2008; Vecchietti et al. 2009; Vizoso et al. 2009) and molecular markers mapped to genetically anchored maps (Joobeur et al. 1998; Wang et al. 2002; Jung et al. 2005). To accomplish the identification of molecular markers that can be associated with higher tolerance to woolliness and to other traits in Prunus species, markers such as RFLP, isozymes (Joobeur et al. 1998), SSR (Yamamoto et al. 2002) and EST-SSR (Howad et al. 2005) has been anchored to a reference map. This reference map is considered key in the development of a selective mapping approach known as Bin mapping (Howad et al. 2005). This approach consist in the positioning of newly developed molecular markers within a previously described genetically anchored map, which in the case of Prunus species belongs to the F₂ backcross of the almond variety Texas with the peach variety Earlygold (Joobeur et al. 1998).

The development of an approach known as intron-flanking EST-PCR markers based on the alignment of EST cDNA sequences against known genomic sequences of Arabidopsis thaliana has been described (Wei et al. 2005). These types of markers are based on the prediction of putative splicing sites and the amplification of them. Considering the assumption that intronic regions are richer in polymorphism than exonic ones, an increment in the use of these markers as fingerprinting tool has been reported for non-model species such as Rhododendron (Wei et al. 2005; De Keyser et al. 2009), Lolium, Festuca (Tamura et al. 2009) and Rosaceae species (Sargent et al. 2009). Taking in consideration the functional association of these type of markers, it could be a good approach in order to search for Quantitative Trait Loci (QTL), which in P. persica has been described already for commercially interesting traits such as soluble sugar and organic acids content (Dirlewanger et al. 1999), fruit weight, skin colour, total soluble solids, juice acidity and juice pH (Eduardo et al. 2011).

The objective of the present work is to design polymorphic markers, using an intron flanking EST-PCR approach. These markers are representative of previously undescribed genes associated to metabolic pathways that could be involved in the triggering of the woolly phenotype. This will allow assigning a position in common regions of the genetically anchored Prunus reference map, which are projected to be useful for future MAS programs.

The genomic DNA used for the screening through the Bin mapping (Howad et al. 2005) approach belongs to the F₂ population of Peach cv. Earlygold X Almond cv. Texas described for the Prunus reference map (TxE) in Joobeur et al. (1998) kindly donated by Dr. Peré Arús and Dr. Werner Howad (IRTA, Spain).

The information describing differentially expressed genes reported in a previous work (Vizoso et al. 2009) was obtained from our database and managed using the JUICE software (Latorre et al. 2006). 32 unigenes were selected for Bin mapping (Table 1). These selection of unigenes previously shown a differential expression when fruits were stored in cold conditions for a prolonged period of time (Vizoso et al. 2009). Based on their ontology, these genes may be related to cell wall metabolism and stress response. From a group of 249 unigenes differentially expressed between harvest and post harvest cold treatment, we select 2 subsets of unigenes according to its Gene Ontology categorization (Harris et al. 2004) for cell wall integrity and stress response. For cell wall integrity a total of 18 genes were selected. The second subset considered 13 genes related with stress response, which in addition to the Hsp70 control makes a total of 32 unigenes (Table 1) used to identify intron flanking EST-PCR molecular markers and position them on the Prunus reference map.

To design molecular markers representative of each candidate gene, we used the intron-flanking PCR molecular marker approach described by Wei et al (2005). This approach consists in a local BLAST-N alignment of the candidate genes against genomic sequences that includes introns and untranslated regions (UTRs) of Arabidopsis thaliana (genome version TAIR10 plus introns and UTRs downloaded from www.arabidopsis.org). Taking in consideration that predictions of putative splicing sites through a comparative analysis have been demonstrate to be highly accurate between A. thaliana and non model species (Wei et al. 2005; De Keyser et al. 2009; Sargent et al. 2009; Tamura et al. 2009) we decide to take this approach for our analysis. In order to design intron flanking PCR molecular markers which will be representative of each candidate gene, the consensus sequence of the assembled contigs obtained from our local database were aligned using BLAST-N against the tenth version of the annotated A. thaliana genome which includes introns and UTRs (http://www.arabidopsis.org/wublast/index2.jsp). This sequence alignment allows putative splicing sites to be predicted (exon-exon joint). Based on the assumption that simple sequence polymorphisms are more abundant in non coding regions, we designed PCR primers that flank exon-exon junction (Figure 1). The primermaster 2.0 program was used for primer design (Proutski and Holmes, 1996). The following criteria were considered: length of primers between 16-24 mer, size of amplicon in cDNA sequence no shorter than 200 bp and no longer than 300 bp, and melting temperature limits between 55ºC - 65ºC. Designed primers and expected size of introns can be observed in Table 2. The contigs included in the molecular marker design were also physically mapped to the Peach 1.0 scaffolds available in the Genome Database for Rosaceae (Jung et al. 2004; Arús et al. 2012) and Phytozome v8.0 (Goodstein et al. 2012). The mapping was done by BLASTX alignment against the Prunus proteome available within these databases (Table 1).

Genotype screening, genetic anchored map position assignment and PCR amplification

To position these candidate genes within the TxE Prunus reference map, we used the Bin mapping approach, described by Howad et al. (2005). The approach consists in a PCR profiling on the 8 most informative plants of the reference map population (2 parental plants and 6 F₂ population plants). The different genetic profile of this small set of plants describes 67 spaces across the 8 linkage groups of the Prunus genetically anchored map (Joobeur et al. 1998), which are determined by the presence of female, male or heterozygous alleles; allele A represents the female parental; B represents the male parental (Earlygold line) and H is the heterozygote (TxE line). PCR amplification of genomic DNA was performed as follow: 25 µL of PCR mixture consisting of 10 ng of genomic DNA; 6.3 µL of nuclease free H₂O; 2.5 µL of 10X RBC® reaction buffer (with 15 mM Mg²⁺); 1 µL of RBC ® 50 mM MgCl₂(to reach a 3.5 mM final Mg²⁺concentration); 1 mM of Invitrogen® dNTPs mixture; 0.5 mM of forward and reverse primers and 1 U of RBC ® Taq DNA Polymerase. The mixtures were incubated for 5 min at 94ºC, followed by 35 cycles of denaturation at 94ºC for 1 min, annealing for 1 min at selected temperatures for each set of primers, and elongation at 72ºC for 1 min, followed by a final extension of 10 min at 72ºC. The PCR amplification products were analyzed by electrophoresis in a 7% acrylamide gel, at 120 V for 2 hrs, and revealed by ethidium bromide staining.

The closest orthologs mapped into the genetically anchored TxE Bin mapping genes were located within the Arabidopsis genome through the chromosome map tool on the TAIR website (http://www.arabidopsis.org). The orthologs names were used as input into the chromosome map and their positions were displayed using the whole genome view.

Alignment gaps were localized and compared to the intron position in Arabidopsis’s sequences for prediction of exon-exon joint putative position within the peach sequences (Figure 1). Primers that flank one or maximum two consecutive introns were designed (Table 2). Additionally, primers that amplify a large fragment within sequences available were designed in those unigenes that present lack of introns in their respective Arabidopsis orthologs (Table 2).

Amplification from genomic DNA was performed on the Binset samples (Figure 2). 72% of the Unigenes analyzed showed evidence of size polymorphism on the Binset TxE population, from which 34% were successfully mapped to a Bin position (Table 3 and Figure 3) and 38% of them showed a polymorphic profile not described in any Bin. The unigene 803 similar to a xyloglucan endotransglycosilase of A. thaliana, and unigene 1686 which is similar to a putative glucose-6-phosphate isomerase from A. thaliana were mapped to the Bin 1:78, which is delimited by RFLP markers BF08A and TubA3. Both unigenes were selected from groups associated with cell wall integrity and carbohydrate metabolism. A group of two candidate genes related with cell wall integrity and carbohydrate metabolism, unigene 2575, similar to a glyceraldehyde-3-phosphate dehydrogenase from Capsicum annuum and unigene 474 that is a Prunus persica phosphoenolpyruvate carboxykinase homolog to one from Solanum lycopersicum, were placed to the Bin 4:18 which is delimited by the RFLP marker BG05A and the SSR marker BPPCT040. The unigene 2131 (from the stress response related group) similar to an endochitinase precursor from A. thaliana was positioned within the Bin 7:48, which is delimited by RFLP marker AG60A and PMS2 SSR marker. In the Bin 7:56 (contiguous to Bin 7:48) was positioned the unigene 1590 (cell wall and carbohydrate metabolism group) which has a domain of a sucrose-UDP glucosyltransferase. The unigene 2785, a hypothetical protein associated to senescence was positioned to the Bin 1:14 delimited by the EST-SSR EPPCU0027 and the SSR UDAp-471. The unigene 1027, similar to the heat shock protein 70 was mapped to the Bin 2:50, delimited by two SSR markers, which are UDA-023 and UDAp-462, both from Prunus amygdalus (Struss et al. 2003). Within the linkage group number 3, just one of the selected candidate genes was mapped to the Bin 3:12, unigene 2957, similar to a protein from the glycosyl hydrolase family 3. The marker designed from the unigene 1879 which showed a weak similitude to the precursor for 4-alpha-glucanotransferase was mapped to Bin 6:39, delimited by the SSR type markers M19a and UDAp-497 from P. persica and P. amygdalus respectively. Within the linkage group 8, a new marker designed from the unigene 2473 sequence, from the cell wall integrity group of candidate genes, was mapped to the Bin 8:41, delimited by the EST-SSR EPPB4226 and the SSR marker MA013a (Yamamoto et al. 2002), both from P. persica. Additionally, the orthologs from A. thaliana of the candidate genes successfully mapped in the Prunus reference map, through the Bin mapping approach, were identified using the TAIR chromosome map (Figure 4).

We did analyze a set of genes potentially involved in abiotic stress mediated by low temperature which also could be involved in cell wall integrity. The criteria used to select genes include those unigenes that in the first place were differentially transcribed after 21 days of cold treatment as described in Vizoso et al. (2009), taking in account that the triggering of the chilling injury is considered to be irreversible after 15 days (Fernández-Trujillo and Artés, 1997). The selection of the candidate genes, based on their GO categories, considered for cell wall integrity the genes that were associated with synthesis or degradation of cell wall and additionally those unigenes with GO category included in any part of the carbohydrate metabolism, since the available pool of carbohydrates could affect the cell wall metabolism (Fischer and Bennett, 1991; Dawson et al. 1992; Trainotti et al. 2003; Brummell et al. 2004). Also those unigenes which were categorized by their GO number within pathways associated with response to abiotic stimuli were also included, taking in account the cross talk between biotic and abiotic responses (Fujita et al. 2006). Therefore an attempt to position genes functionally associated to chilling injury in the Prunus reference map was made.

As previously has been described, the intron flanking approach showed to be a fast and efficient methodology to design polymorphic markers (Wei et al. 2005; De Keyser et al. 2009; Sargent et al. 2009; Tamura et al. 2009). This approach takes advantage of the Arabidopsis reference genome to predict potential polymorphic sites within candidate genes from different species. In the case of this study showed a 72% of polymorphic markers within the Binset population of the TxE Prunus reference map designed with this strategy. These results could be explained taking in consideration that the Bin mapping approach does not cover the whole TxE Prunus reference map, and the polymorphic profiles found for the non positioned markers could be associated to regions within the genetic map uncovered by the Bin mapping approach. On the other hand, from the successfully positioned genes, 2 pairs of genes were mapped to a same Bin. The first case was found in the Bin 1:78 that have a xyloglucan endotransglycosilase and a putative glucose-6-phosphate isomerase within its 2.8 cM genetic distance. In the second case, the genes glyceraldehyde-3-phosphate dehydrogenase and a unigene similar to a phosphoenolpyruvate carboxykinase were mapped together to the Bin 4:18. Additionally a similar result was found for the unigene pair composed by unigene 2131 similar to an endochitinase which was mapped to the Bin 7:48 adjacent to the Bin 7:56 where was mapped the unigene 1590 similar to a sucrose-UDP glucosyltransferase. This could indicate that these regions in the Prunus reference map may be potential regulations zones for genes associated with carbohydrate metabolism, cell wall integrity control and stress response. In the scaffold 1 of the Prunus v1.0 were positioned 12 unigenes (38%), 7 of them belonging to the cell wall and carbohydrate metabolism group and 5 of them from the stress response group of genes, suggesting that the genes involved in the regulation of these pathways can be clustered within this Chromosome. Therefore these genes could be suggested to be used as potentially markers in a molecular assisted breeding program.

The approach used in this work appears to be highly effective to design a simple and informative type of molecular marker. These markers can be used to address the role of selected genes against a stress condition. According to the percentage of successfully mapped genes, we suggest that the average position accuracy is directly related to the low number of candidates assigned to a genetic map position. When compared the position of the closest orthologs to the Arabidopsis genome, it can be observed a dispersed distribution of them on the positioning, similar to what has been reported in previous studies (Dominguez et al. 2003; Georgi et al. 2003; Jung et al. 2006; Sargent et al. 2009). However the homology at the transcript level between P. persica and A. thaliana has been shown to be informative for the prediction of putative splicing sites which can be used as intron flanking tool. This homology have been observed as well in a previous study (Tittarelli et al. 2009) to be conserved in the transcription regulation between these two species.

Recently, new molecular findings related to chilling injury traits have been described (Cantín et al. 2010; Martínez-García et al. 2012) therefore the molecular markers described in our work are a new set that can be useful for a Molecular Assisted Selection program. The intron flanking EST-PCR markers described in this work as well as new ones are projected to be used in the evaluation of different peach genotypes as well as segregating populations for traits related to chilling injury.

The majority of the approaches that look for molecular markers associated with biologically relevant phenotypes search for a pattern that can be related to a trait segregating within a large population. The methodology used in this work melt several approaches in search for the positioning of markers designed on the base of functionally linked genes. The in silico profiling of candidate genes obtained from cDNA libraries of mesocarp tissue from different post harvest condition treatments allowed us to assign a position on the Prunus reference map to a group of new genes that also were differentially transcribed under cold treatment. The search for putative polymorphisms in non-coding regions within these genes is a very effective way to identify molecular markers representative of specific candidate genes, which could be useful for screening segregating populations. This, in addition to the Bin mapping approach makes a fast and efficient way to choose regions that could play an important role in the triggering of differential gene expression under a stress condition. Nevertheless for this last assumption an evaluation of segregant populations of P. persica is needed. As a final remark, it is important to mention that the results obtained in this work are intended to be used in Molecular Assisted Selections programs for the development of new cultivars with good post harvest behaviour under chilling injury conditions.

We thank the kind collaboration of Dr. Peré Arús and Dr. Werner Howad from IRTA Barcelona, Spain who provided us with the genomic DNA from the Binset population. SDM is a member of the Biotechnology Ph.D. Program at Universidad Andres Bello.

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