Luca Dondini

The Peach v2.0 release: high-resolution linkage mapping and deep resequencing improve chromosome-scale assembly and contiguity

BackgroundThe availability of the peach genome sequence has fostered relevant research in peach and related Prunus species enabling the identification of genes underlying important horticultural traits as well as the development of advanced tools for genetic and genomic analyses. The first release of the peach genome (Peach v1.0) represented a high-quality WGS (Whole Genome Shotgun) chromosome-scale assembly with high contiguity (contig L50 214.2xa0kb), large portions of mapped sequences (96%) and high base accuracy (99.96%). The aim of this work was to improve the quality of the first assembly by increasing the portion of mapped and oriented sequences, correcting misassemblies and improving the contiguity and base accuracy using high-throughput linkage mapping and deep resequencing approaches.ResultsFour linkage maps with 3,576 molecular markers were used to improve the portion of mapped and oriented sequences (from 96.0% and 85.6% of Peach v1.0 to 99.2% and 98.2% of v2.0, respectively) and enabled a more detailed identification of discernible misassemblies (10.4xa0Mb in total). The deep resequencing approach fixed 859 homozygous SNPs (Single Nucleotide Polymorphisms) and 1347 homozygous indels. Moreover, the assembled NGS contigs enabled the closing of 212 gaps with an improvement in the contig L50 of 19.2%.ConclusionsThe improved high quality peach genome assembly (Peach v2.0) represents a valuable tool for the analysis of the genetic diversity, domestication, and as a vehicle for genetic improvement of peach and related Prunus species. Moreover, the important phylogenetic position of peach and the absence of recent whole genome duplication (WGD) events make peach a pivotal species for comparative genomics studies aiming at elucidating plant speciation and diversification processes.

Genome-Wide Association Mapping of Flowering and Ripening Periods in Apple

Deciphering the genetic control of flowering and ripening periods in apple is essential for breeding cultivars adapted to their growing environments. We implemented a large Genome-Wide Association Study (GWAS) at the European level using an association panel of 1,168 different apple genotypes distributed over six locations and phenotyped for these phenological traits. The panel was genotyped at a high-density of SNPs using the Axiom®Apple 480 K SNP array. We ran GWAS with a multi-locus mixed model (MLMM), which handles the putatively confounding effect of significant SNPs elsewhere on the genome. Genomic regions were further investigated to reveal candidate genes responsible for the phenotypic variation. At the whole population level, GWAS retained two SNPs as cofactors on chromosome 9 for flowering period, and six for ripening period (four on chromosome 3, one on chromosome 10 and one on chromosome 16) which, together accounted for 8.9 and 17.2% of the phenotypic variance, respectively. For both traits, SNPs in weak linkage disequilibrium were detected nearby, thus suggesting the existence of allelic heterogeneity. The geographic origins and relationships of apple cultivars accounted for large parts of the phenotypic variation. Variation in genotypic frequency of the SNPs associated with the two traits was connected to the geographic origin of the genotypes (grouped as North+East, West and South Europe), and indicated differential selection in different growing environments. Genes encoding transcription factors containing either NAC or MADS domains were identified as major candidates within the small confidence intervals computed for the associated genomic regions. A strong microsynteny between apple and peach was revealed in all the four confidence interval regions. This study shows how association genetics can unravel the genetic control of important horticultural traits in apple, as well as reduce the confidence intervals of the associated regions identified by linkage mapping approaches. Our findings can be used for the improvement of apple through marker-assisted breeding strategies that take advantage of the accumulating additive effects of the identified SNPs.

Comparative Analysis of SSR Markers Developed in Exon, Intron, and Intergenic Regions and Distributed in Regions Controlling Fruit Quality Traits in Prunus Species: Genetic Diversity and Association Studies

Simple sequence repeats (SSRs) are genome domains located in both coding and non-coding regions in eukaryotic genomes. Although SSRs are often characterized by low polymorphism, their DNA-flanking sequences could be a useful source of DNA markers, which could help in genetic studies and breeding because they are associated with genes that control traits of interest. In this study, 56 genotypes from different Prunus species were used, including peach, apricot, plum, and almond (already phenotyped for several agronomical traits, including self-compatibility, flowering and ripening time, fruit type, skin and flesh color, and shell hardness). These Prunus genotypes were molecularly characterized using 28 SSR markers developed in exons, introns, and intergenic regions. All these genes were located in specific regions where quantitative trait loci (QTLs) for certain fruit quality traits were also located, including flowering and ripening times and fruit flesh and skin color. A sum of 309 SSR alleles were identified in the whole panel of analyzed cultivars, with expected heterozygosity values of 0.61 (upstream SSRs), 0.17 (exonic SSRs), 0.65 (intronic SSRs), and 0.58 (downstream SSRs). These values prove the low level of polymorphism of the exonic (gene-coding regions) markers. Cluster and structural analysis based on SSR data clearly differentiated the genotypes according to either specie (for the four species) and pedigree (apricot) or geographic origin (Japanese plum). In addition, some SSR markers mainly developed in intergenic regions could be associated with genes that control traits of interest in breeding and could therefore help in marker-assisted breeding. These findings highlight the importance of using molecular markers able to discriminate between the functional roles of the gene allelic variants.

Characterization of 25 full-length S-RNase alleles, including flanking regions, from a pool of resequenced apple cultivars

Key messageData obtained from Illumina resequencing of 63 apple cultivars were used to obtain full-length S-RNase sequences using a strategy based on both alignment and de novo assembly of reads.AbstractThe reproductive biology of apple is regulated by the S-RNase-based gametophytic self-incompatibility system, that is genetically controlled by the single, multi-genic and multi-allelic S locus. Resequencing of apple cultivars provided a huge amount of genetic data, that can be aligned to the reference genome in order to characterize variation to a genome-wide level. However, this approach is not immediately adaptable to the S-locus, due to some peculiar features such as the high degree of polymorphism, lack of colinearity between haplotypes and extensive presence of repetitive elements. In this study we describe a dedicated procedure aimed at characterizing S-RNase alleles from resequenced cultivars. The S-genotype of 63 apple accessions is reported; the full length coding sequence was determined for the 25xa0S-RNase alleles present in the 63 resequenced cultivars; these included 10 previously incomplete sequences (S5, S6a, S6b, S8, S11, S23, S39, S46, S50 and S58). Moreover, sequence divergence clearly suggests that alleles S6a and S6b, proposed to be neutral variants of the same alleles, should be instead considered different specificities. The promoter sequences have also been analyzed, highlighting regions of homology conserved among all the alleles.

QTLs for susceptibility to Stemphylium vesicarium in pear

Brown spot is one of the most serious fungal diseases that can affect pear fruits and leaves in the Po valley (Italy). Stemphylium vesicarium is the causal agent of this disease, and several antifungal treatments, repeated throughout the period between bloom and harvest, are needed to control its spread. Many of the most important pear cultivars (such as ‘Abbé Fétel’) are very susceptible to this fungus, while others (such as ‘Bartlett’ and its mutated sports) are known to be resistant. Our research aimed to develop molecular markers linked to this trait. To this end, 92 seedlings derived from an ‘Abbé Fétel’u2009×u2009‘Max Red Bartlett’ cross were evaluated for resistance to S. vesicarium for two consecutive years by artificial inoculation with conidia on detached leaves and fruits under controlled conditions in greenhouse. The extent of the lesions was recorded at different time points. A major QTL for susceptibility was located at the lower end of linkage group 15 of ‘Abbé Fétel’. This region was saturated with three SSR markers, and the putative position of a susceptibility gene was also estimated by the single gene mapping approach. This putative gene was located at 2xa0cM far from the lower end of the linkage group. Molecular markers tightly associated to this locus represent a first step towards the development of MAS (marker-assisted selection) to support the selection of new pear genotypes more resistant to brown spot.

Seed morphometry is suitable for apple-germplasm diversity-analyses

Abstract The main objective of this study was to evaluate the trustworthiness of seed image analysis as an approach to discriminate apple germplasm accessions. Digital images of seeds from 42 apple cultivars, acquired by a flatbed scanner, provided a phenotypic dataset with 106 morphometric variables. Stepwise Linear Discriminant Analysis (LDA) was used to examine this dataset, and the results were compared with available genetic data. The first comparison among cultivars provided a 38.8% cross-validation of correct identifications with a discriminant percentage ranging between 11.7 and 70%. In agreement with the genetic diversity analysis, the LDA could discriminate between the apples cultivars, identifying two main groups that could be further divided into additional subgroups. Based on our findings, we propose that seed image analysis is a valuable and affordable tool to investigate phenotypic diversity among a large number of apple cultivars.