FRUIT WEIGHT IN PEACH: ASSESSING THE GENETIC POTENTIAL THROUGH PHENOTYPIC AND GENOMIC TOLLS

The trait fruit weight is of great agronomic importance for the commercial production of peach. In view of conducting a study of association mapping, the genetic diversity of peach accessions from the germplasm bank of MAS.PES was evaluated using amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR). The results indicated that SSR markers were more informative and showed a high level of homozygosity in the accessions under evaluation. In studies meant for breeding of this species, the identification of quantitative trait loci (QTLs) explaining the phenotypic differences of this trait is important, because they can be used as a genomic tool in marker-assisted selection (MAS). In the present study, QTLs were detected through two approaches: 1. Linkage analysis, starting from a F2 population of 123 individuals from NJ Weeping (small fruit) x Bounty (large fruit), in which the software JoinMap was used to establish the relationship of linkage between the segregating markers, while the software MapQTL was employed to associate the data with the obtained phenotypic linkage map. 2. Association mapping, carried out on 70 peach accessions from the MAS.PES germplasm collection, in which the software Structure was used to analyse population structure: associations between markers and phenotypic traits were identified with the software TASSEL. As a result of linkage analysis, 877 single-nucleotide polymorphism (SNP) markers were grouped into 8 linkage groups and 34 QTLs related to fruit weight and size were identified. The results of the association analysis, performed using the General Linear Model (GLM), suggested the existence of 39 markers associated with fruit weight and size

Genetic Dissection of Complex Fruit Quantitative Traits in Peach Progen

Major research efforts in peach are dedicated to the discovery of genomic variants causing phenotypic effects in complex fruit traits such as: maturity date (MD), fruit size (FW), sugar (SSC) and acid content (TA), flesh texture (slow softening, SSf) and resistance to brown rot by Monilinia spp. (BRr). Five segregating progenies showing phenotypic variation for at least one of these traits are available in our experimental fields. For SSC and TA, an already validated approach based on Near-InfraRed spectroscopy (NIR), is being applied to phenotype some segregating progenies. For SSf and BRr instead, trait characterization has been performed, resulting in the identification of co-factor traits and definition of standardized phenotyping tools, which are currently applied in the characterization of segregating material (in the context of FruitBreedomics EU project). High-density linkage maps have been constructed with genotypic data obtained from IPSC Illumina 9K SNP chip (Italian Drupomics and FruitBreedomics frameworks) and Genotyping-by-Sequencing (GBS). Additionally, parents of these progenies have been re-sequenced (30-40x) and genetic variants present along their genomes have been identified. Multiple-QTL models (MQM) coupled with the use of co-factor traits is leading to the discovery of significant QTLs. Genomic variants are explored within QTL intervals on the genomes of progeny parents, in order to identify possible mutations causing phenotypic differences, and develop markers for marker-assisted selection approaches

Development and Evaluation of a 9K SNP Addition to the Peach Ipsc 9K SNP Array v1

The IPSC 9K peach SNP array released by the international community has been a valuable tool in research and application. Even though majority of SNPs (84%) were polymorphic in the evaluation panels there were many genomic regions with low coverage, including those important for breeding. The existing peach array has been updated with 9K additional SNPs covering previously identified gaps and including recently identified SNPs important for breeding. SNPs (1,808,996) identified by sequencing 49 genomes of additional peach accessions were used as the main source of additional SNPs. Focal point strategy was used to select 8,971 SNPs within 40kb window from the 2,821 focal points distributed across the genome. Additional 129 SNPs were chosen to saturate either regions important for breeding or close the gaps larger than 100kb. The array was validated with 1,770 peach and 26 Prunus accessions (almond, plum, apricot, wild relatives). The add-on contained 7,862 SNPs evenly spread across 8 peach pseudo-molecules with only one SNP positioned on scaffold 13 covering 224.99Mbp of peach genome. The 9K add-on improved the 9K peach array by increasing the total number of usable SNPs by 7,206. The number of SNPs per chromosome increased on average by 50% with only on average 0.18% increase in total physical coverage. Number of gaps larger than 0.3 Mbp was reduced to 2 one on each chromosome 3 and 8. Overall genotyping efficiency in all material was >90% except in almond, 82%. Number of informative markers, assessed by ASSIsT software, were highest in peach 64% and lowest in almond 10%, with 61% of markers being informative in wild Prunus (12) and 35% in apricot (4) and 2 - 33% in Japanese and European plum, respectively. Among 36.2% discarded markers 33% were monomorphic and 30% shifted homozygous in material used. Those markers could be informative in different background raising total number of informative markers. Ann addition of new SNPs to array improved the density and usefulness of the array in Prunus species. The practical applications of new 16K Illumina SNP peach array will be discussed. Specified Source(s) of Funding: USDA-NIFA-SCRI-Ros- BREED (2014-51181-22378

Deletion of the miR172 target site in a TOE-type gene is a strong candidate variant for dominant double-flower trait in Rosaceae

Double flowers with supernumerary petals have been selected by humans for their attractive appearance and commercial value in several ornamental plants, including Prunus persica (peach), a recognized model for Rosaceae genetics and genomics. Despite the relevance of this trait, knowledge of the underlying genes is limited. Of two distinct loci controlling the double-flower phenotype in peach, we focused on the dominant Di2 locus. High-resolution linkage mapping in five segregating progenies delimited Di2 to an interval spanning 150858bp and 22 genes, including Prupe.6G242400 encoding an euAP2 transcription factor. Analyzing genomic resequencing data from single- and double-flower accessions, we identified a deletion spanning the binding site for miR172 in Prupe.6G242400 as a candidate variant for the double-flower trait, and we showed transcript expression for both wild-type and deleted alleles. Consistent with the proposed role in controlling petal number, Prupe.6G242400 is expressed in buds at critical times for floral development. The indelDi2 molecular marker designed on this sequence variant co-segregated with the phenotype in 621 progenies, accounting for the dominant inheritance of the Di2 locus. Further corroborating the results in peach, we identified a distinct but similar mutation in the ortholog of Prupe.6G242400 in double-flower roses. Phylogenetic analysis showed that these two genes belong to a TARGET OF EAT (TOE)-type clade not represented in Arabidopsis, indicating a divergence of gene functions between AP2-type and TOE-type factors in Arabidopsis and other species. The identification of orthologous candidate genes for the double-flower phenotype in two important Rosaceae species provides valuable information to understand the genetic control of this trait in other major ornamental plants. Significance Statement We used peach as a model to gain insight into the molecular basis of double flowers, an important trait in many ornamental plants. We propose that a deletion causes a TOE-type transcription factor to escape miR172-mediated repression, in turn resulting in an increased number of petals, as corroborated by results on the orthologous gene in rose

High-density multi-population consensus genetic linkage map for peach

Highly saturated genetic linkage maps are extremely helpful to breeders and are an essential prerequisite for many biological applications such as the identification of marker-trait associations, mapping quantitative trait loci (QTL), candidate gene identification, development of molecular markers for marker-assisted selection (MAS) and comparative genetic studies. Several high-density genetic maps, constructed using the 9K SNP peach array, are available for peach. However, each of these maps is based on a single mapping population and has limited use for QTL discovery and comparative studies. A consensus genetic linkage map developed from multiple populations provides not only a higher marker density and a greater genome coverage when compared to the individual maps, but also serves as a valuable tool for estimating genetic positions of unmapped markers. In this study, a previously developed linkage map from the cross between two peach cultivars 'Zin Dai' and 'Crimson Lady' (ZC2) was improved by genotyping additional progenies. In addition, a peach consensus map was developed based on the combination of the improved ZC2 genetic linkage map with three existing high-density genetic maps of peach and a reference map of Prunus. A total of 1,476 SNPs representing 351 unique marker positions were mapped across eight linkage groups on the ZC2 genetic map. The ZC2 linkage map spans 483.3 cM with an average distance between markers of 1.38 cM/marker. The MergeMap and LPmerge tools were used for the construction of a consensus map based on markers shared across five genetic linkage maps. The consensus linkage map contains a total of 3,092 molecular markers, consisting of 2,975 SNPs, 116 SSRs and 1 morphological marker associated with slow ripening in peach (SR). The consensus map provides valuable information on marker order and genetic position for QTL identification in peach and other genetic studies within Prunus and Rosaceae

Solution Structure and Phylogenetics of Prod1, a Member of the Three-Finger Protein Superfamily Implicated in Salamander Limb Regeneration

Prod1 is a cell-surface molecule of the three-finger protein (TFP) superfamily involved in the specification of newt limb PD identity. The TFP superfamily is a highly diverse group of metazoan proteins that includes snake venom toxins, mammalian transmembrane receptors and miscellaneous signaling molecules..The available data suggest that Prod1, and thereby its role in encoding PD identity, is restricted to salamanders. The lack of comparable limb-regenerative capability in other adult vertebrates could be correlated with the absence of the Prod1 gene

Genetic variability and population structure of peach accessions from MAS.PES germplasm bank

The aim of the present study was to analyse the genetic diversity and determine the population structure and the phylogenetic relationships of 123 peach accessions from the germplasm bank of the \u2018MAS.PES\u2019 breeding programme in order to support future research on breeding activities. Fifteen SSR markers were selected on the basis of a previous study showing a high level of allelic diversity. The population structure was analysed using the software STRUCTURE, adopting the admixture model of ancestry, and the phylogenetic analysis was performed by the DARWIN software, using the neighbour-joining method. The three clusters obtained by analysis of population structure were in accordance with the analysis performed by the software DARWIN. The fifteen studied SSRs amplified 115 alleles, with an average of 7.7 alleles per locus. The most informative markers were BPPCT001, UDP98-412 and EPPCU5176. The observed heterozygosity demonstrates an average of 0.4 alleles per locus, lower than the expected heterozygosity. The fixation index (F) values were positive in all loci, with an average of 0.3 alleles per locus. This result confirms the presence of inbreeding in the evaluated accession, as commonly found in cultivated peach

Identifying SNP markers tightly associated with six major genes in peach [Prunus persica (L.) Batsch] using a high-density SNP array with an objective of marker-assisted selection (MAS)

One of the applications of genomics is to identify genetic markers linked to loci responsible for variation in phenotypic traits, which could be used in breeding programs to select individuals with favorable alleles, particularly at the seedling stage. With this aim, in the framework of the European project FruitBreedomics, we selected five main peach fruit characters and a resistance trait, controlled by major genes with Mendelian inheritance: fruit flesh color Y, fruit skin pubescence G, fruit shape S, sub-acid fruit D, stone adhesion-flesh texture F-M, and resistance to green peach aphid Rm2. They were all previously mapped in Prunus. We then selected three F1 and three F2 progenies segregating for these characters and developed genetic maps of the linkage groups including the major genes, using the single nucleotide polymorphism (SNP) genome-wide scans obtained with the International Peach SNP Consortium (IPSC) 9K SNP array v1. We identified SNPs co-segregating with the characters in all cases. Their positions were in agreement with the known positions of the major genes. The number of SNPs linked to each of these, as well as the size of the physical regions encompassing them, varied depending on the maps. As a result, the number of useful SNPs for marker-assisted selection varied accordingly. As a whole, this study establishes a sound basis for further development of MAS on these characters. Additionally, we also discussed some limitations that were observed regarding the SNP array efficiency

Exploring and exploiting phenotypic and genetic diversity in peach: identification of major genes and QTLs by GWAS

Genetic variability is a key requirement for breeding. Although new peach cultivars are released yearly to the market, the genetic pool of cultivated peaches is very limited. To evaluate the variability available in commercial but also in old local peach accessions we selected a panel of 1,580 accessions maintained and evaluated in four European and one Chinese germplasm collections. Phenotypic data collected over years following common protocols have been integrated in a database, generating a useful tool for breeders and researchers. These accessions were genotyped with the peach 9K SNP array v1. T. Genotypic data distributed the accessions in three main subpopulations (Occidental obtained in breeding programs, Occidental old local varieties and Chinese cultivars). Linkage disequilibrium (LD) was in agreement with previous studies reporting long extension. Phenotypic and genotypic data have been combined in a GWAS study allowing the design of markers for marker assisted selection (MAS). Preliminary analyses on quantitative traits are promising, while further analysis will be required to integrate all data in a single genome-wide association analysis