Identification of a new allele of the Dw gene causing brachytic dwarfing in peach
Peach brachytic dwarfism determined by Dwarf gene (Dw) is an undesired trait segregating in some peach breeding programs. Recently, a single nucleotide polymorphism (SNP) mutation in the gibberellin insensitive dwarf 1 (GID1) peach gene causing brachytic dwarfism was described. In this research we wanted to validate this marker in an F2 population of the ‘Nectavantop’ peach cultivar (Nv) to include it as a marker assisted selection tool for peach breeding programs.
The observed segregation of the trait was in agreement with that of a recessive gene, the individuals homozygous for the recessive allele (dwdw) presenting the dwarf genotype. Dw was mapped to the distal part of linkage group 6 as previously described. The SNP marker based on the causal mutation previously described did not segregate in Nv F2 population. The sequence of the GID1c gene in Nv revealed a second SNP in its coding sequence which cosegregated with the dwarf phenotype. This SNP was predicted by the SNAP2 software to cause a major functional change and was validated in the dwarf peach cultivar ‘Small sunning’. These results suggest the existence of at least two independent mutations of the Dw gene causing the peach brachytic dwarf phenotype.
KeywordsTree architecture Molecular marker Prunus persica Marker-assisted selection
gibberellin insensitive dwarf 1
marker assisted introgression
polymerase chain reaction
single nucleotide polymorphism
simple sequence repeat
Until today different dwarf peach phenotypes have been described and different applications have been proposed for them, including the development of dwarf cultivars for intensive production, their use as ornamentals, and the use of genetically modified cultivars for the dwarfing genes to control fruit architecture . The genetics of dwarf phenotypes has been studied and several major genes (Dw, Dw2, Dw3) have been identified [2, 3, 4, 5, 6]. Homozygous individuals for either dw or dw2 display brachytic dwarfism (BD), presenting short internodes, thickened stems, reduced higher order branching, elongated leaves and normal fruit. Homozygous individuals for dw3 are different from the previous ones and present narrow branches and willowy growth . Dwarfing was also determined by a fourth gene (N) where the nn homozygote has short internodes, but Nn heterozygotes generate semi-dwarf individuals [8, 9]. Another dwarfing phenotype, recently described by Lu et al. , is the temperature-sensitive semi-dwarf (Tssd) locus that regulates internode length. The presence of the dominant Tssd allele determines short internode length at temperatures below 30 °C. Dw and Tssd have been mapped to the distal part of chromosome 6 [6, 11] and to the proximal part of chromosome 3 , respectively. Only Dw has been cloned , being the dw allele generated by a non-sense mutation resulting in a non-functional product of the gibberellic acid (GA) receptor PpeGID1c gene . PpeGID1c is annotated as Prupe.6G332800 in the peach genome v2.0 and as ppa018174 in v1.0. For the other dwarfing genes there is no information on their map positions or markers tightly linked to them.
BD individuals are often found to be segregating in peach breeding programs and they are usually discarded. Molecular markers such as the gid1c SNP described by Hollender et al. , can be used as tools for early identification and removal of dwarf plants in peach breeding programs or to avoid crosses between carriers of the dw allele. Several molecular markers for other traits such as fruit shape , fruit acidity  and the slow ripening phenotype , among others , are already being used routinely for marker-assisted selection (MAS) in peach . In this work, we describe the identification of a new allele from the Dw gene producing dwarf individuals found in ‘Nectavantop’ and ‘Small sunning’ cultivars.
Plant material and DNA extraction
A dwarf segregating population of 77 individuals obtained from the open pollination (OP) of ‘Nectavantop’ (Nv) cultivar was used for this study. Trees were planted on their own roots in 2013 in the plots at the IRTA Experimental Station at Gimenells (Lleida, Spain). Trees were visually classified as normal or dwarf during 2 consecutive years (2014 and 2015), that corresponded to the first and second year after planting. Concurrently, one tree of dwarf cultivars ‘Bonanza’ (Bo) and ‘Small sunning’ (Ss) were grown in 50 L pots in the IRTA greenhouse at Torre Marimon (Caldes de Montbui, Spain). DNA from the 77 genotypes of Nv⊗ and Nv, Bo and Ss was extracted from young leaf tissue using the Doyle and Doyle  protocol adapted to 96 well plates.
Primers of the five new SSRs used to map the dwarf trait and the two primers for SNP genotyping
Nv⊗ was first screened with four SSR markers (UDP98-412, MA014a, CPPCT030, CPPCT021) known to be located at the distal end of linkage group 6 (G6) where the peach Dw gene had been previously mapped [6, 11]. Five additional SSRs were developed at this region using the peach genome sequence v2.0 (http://www.rosaceae.org), and the set of SSRs described in http://services.appliedgenomics.org/projects/drupomics/gbrowse/. The new SSRs were noted as CPP followed by the number assigned to this SSR in the IGA (Istituto di Genomica Applicata) annotation of the peach genome v1.0 (Table 1). Six out of the nine SSRs screened in Nv were heterozygous and were genotyped in the progeny. The other three SSRs (MA014a, CPPCT021 and CPP24554) were homozygous in Nv and could not be used for mapping. Eight individuals were found to contain alleles different from those of Nv in at least one marker indicating that they came from cross pollination and were discarded from the dataset. The remaining 69 individuals were considered as true F2 individuals and used for map construction.
A linkage map was constructed using JoinMap v.4.1  software. Groups were established with a LOD ≥ 3.0 and the map was calculated with the Kosambi distance function. Linkage group nomenclature follows the Prunus reference map (T × E) . MapChart 2.1 software  was used to draw the map. To predict the effects of a new sequence variant identified in this research on the function of the PpeGID1c gene product we used the SNAP2 software program .
Genetic mapping of Dw
SNP identification in the GID1c candidate gene
SNP effect prediction
The new SNP-S178F results in an amino acid substitution of a serine (TCT) for a phenylalanine (TTT) in the gibberellic acid receptor protein encoded by the GID1c gene. To predict the possible effect of this mutation in the product of this gene, its sequence was analyzed with SNAP2 software. The SNP-S178F is located in a region of 12 amino acids where mutations can have a high effect on the protein functionality. In our case, the substitution of a Serine by a Phenylalanine presents a high score only exceeded by three other amino acidic changes (Additional file 1), suggesting that the effect of this mutation is sufficiently important to disrupt the functionality of the transcribed protein with phenotypic effects that are indistinguishable from the allele detected by the SNP-W162*, originally found in the Japanese cultivar ‘Juseito’, which produces a stop codon .
Our results indicate that the BD phenotype segregating in the Nv F2 population, although mapping in the same position at the distal part of G6, is not caused by the SNP described by Hollender et al. , but by a different allele. As it occurs for other peach genes that have been characterized at the molecular level in peach, such as the white vs. yellow flesh color , various alleles of different origin may cause similar recessive phenotypes. One of the consequences of this is that selection for markers based on the causal allele may be useful for only one transect of the variability of the species and are useless for the other. Knowledge of the pedigree of the individuals sampled is then a requirement for the adequate choice of markers. This shows also that even in a species with a low level of variability like peach , multiallelic series may be common for genes with phenotypic effects. While in the case of the yellow vs. white gene may be attributed to the amplification of variability caused by human selection for characters related with the edible part of the plant in crop species, it appears not to be the case for the dwarf gene, where the homozygous recessive dwarf individuals are usually rejected.
The main limitations of our results are the low resolution of our mapping population, probably because of the reduced number of individuals used and the low number of cultivars where both SNPs markers, W162* and S178F, could be validated.
CC provided plant material, phenotypic data and helped to draft the manuscript. PA participated in the design of the study and helped to draft the manuscript. IE performed the data analysis, conceived the study and participated in the design and coordination, and drafted the manuscript. All authors read and approved the manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
We acknowledge financial support from the Spanish Ministry of Economy and Competitiveness, through the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016–2019 (SEV‐2015‐0533)” and project AGL2012-40228, and from the CERCA Programme from the Generalitat de Catalunya.
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