Jorge Urrestarazu

Genetic diversity and structure of local apple cultivars from Northeastern Spain assessed by microsatellite markers

A set of 493 old and local Spanish accessions of apple (Malus x domestica Borkh) maintained at three collections in Northeastern Spain was studied using 16 simple sequence repeats in order to estimate their genetic diversity and to identify the genetic structure and relationships among their accessions. An additional diverse set of 45 apple cultivars, including old Spanish and international cultivars, was added as reference. Genetic analyses performed by Bayesian model-based clustering revealed a very strong differentiation of two major groups. The first one clustered 159 individuals (52xa0% of unique genotypes) including local accessions and six old Spanish cultivars. The second major group was formed by 145 individuals, including 38 international reference cultivars and one old Spanish cultivar. Nested Bayesian clustering was applied to those two groups and two and four sub-groups were found at each one, respectively. The identification of private and unique alleles, and the remarkable differences in allelic richness among groups and sub-groups constitute further evidence of a clear genetic structure. The results obtained through the factorial correspondence and analyses of molecular variance confirmed those obtained by Bayesian analyses, revealing moderate but significant differentiation among the two major groups (FSTu2009=u20090.076) and the six sub-groups (FSTu2009=u20090.111). Our results highlight that the genetic diversity encompassed by currently cultivated apple accounts only for a small fraction of that existing within the species, and that an important part (≈60xa0%) of the local material analyzed constitutes a good example of genetic distinctness with respect to the main cultivars used in European orchards.

Analysis of the genetic diversity and structure across a wide range of germplasm reveals prominent gene flow in apple at the European level.

BackgroundThe amount and structure of genetic diversity in dessert apple germplasm conserved at a European level is mostly unknown, since all diversity studies conducted in Europe until now have been performed on regional or national collections. Here, we applied a common set of 16 SSR markers to genotype more than 2,400 accessions across 14 collections representing three broad European geographic regions (Northu2009+u2009East, West and South) with the aim to analyze the extent, distribution and structure of variation in the apple genetic resources in Europe.ResultsA Bayesian model-based clustering approach showed that diversity was organized in three groups, although these were only moderately differentiated (FSTu2009=u20090.031). A nested Bayesian clustering approach allowed identification of subgroups which revealed internal patterns of substructure within the groups, allowing a finer delineation of the variation into eight subgroups (FSTu2009=u20090.044). The first level of stratification revealed an asymmetric division of the germplasm among the three groups, and a clear association was found with the geographical regions of origin of the cultivars. The substructure revealed clear partitioning of genetic groups among countries, but also interesting associations between subgroups and breeding purposes of recent cultivars or particular usage such as cider production. Additional parentage analyses allowed us to identify both putative parents of more than 40 old and/or local cultivars giving interesting insights in the pedigree of some emblematic cultivars.ConclusionsThe variation found at group and subgroup levels may reflect a combination of historical processes of migration/selection and adaptive factors to diverse agricultural environments that, together with genetic drift, have resulted in extensive genetic variation but limited population structure. The European dessert apple germplasm represents an important source of genetic diversity with a strong historical and patrimonial value. The present work thus constitutes a decisive step in the field of conservation genetics. Moreover, the obtained data can be used for defining a European apple core collection useful for further identification of genomic regions associated with commercially important horticultural traits in apple through genome-wide association studies.

Evaluating the Influence of the Microsatellite Marker Set on the Genetic Structure Inferred in Pyrus communis L.

Fingerprinting information can be used to elucidate in a robust manner the genetic structure of germplasm collections, allowing a more rational and fine assessment of genetic resources. Bayesian model-based approaches are nowadays majorly preferred to infer genetic structure, but it is still largely unresolved how marker sets should be built in order to obtain a robust inference. The objective was to evaluate, in Pyrus germplasm collections, the influence of the SSR marker set size on the genetic structure inferred, also evaluating the influence of the criterion used to select those markers. Inferences were performed considering an increasing number of SSR markers that ranged from just two up to 25, incorporated one at a time into the analysis. The influence of the number of SSR markers used was evaluated comparing the number of populations and the strength of the signal detected, and also the similarity of the genotype assignments to populations between analyses. In order to test if those results were influenced by the criterion used to select the SSRs, several choosing scenarios based on the discrimination power or the fixation index values of the SSRs were tested. Our results indicate that population structure could be inferred accurately once a certain SSR number threshold was reached, which depended on the underlying structure within the genotypes, but the method used to select the markers included on each set appeared not to be very relevant. The minimum number of SSRs required to provide robust structure inferences and adequate measurements of the differentiation, even when low differentiation levels exist within populations, was proved similar to that of the complete list of recommended markers for fingerprinting. When a SSR set size similar to the minimum marker sets recommended for fingerprinting it is used, only major divisions or moderate (F ST>0.05) differentiation of the germplasm are detected.

Analysis of the genetic diversity and structure of the Spanish apple genetic resources suggests the existence of an Iberian genepool

87 Pags.- 3 Tabls.- 5 Figs.- 1 Suppl. Fig.- 5 Suppl. Tabls. The definitive version is available at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1744-7348

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.

Genetic diversity of Spanish Prunus domestica L. germplasm reveals a complex genetic structure underlying

European plum (Prunus domestica L.) is an ancient domesticated species cultivated in temperate areas worldwide whose genetic structure has been scarcely analyzed to date. In this study, a broad representation of Spanish European plum germplasm collected in Northeastern Spain and a representative set of reference cultivars were compared using nuclear and chloroplast markers. The number of alleles per locus detected with the SSR markers ranged from 8 to 39, with an average of 23.4 alleles, and 8 haplotypes were identified. Bayesian model-based clustering, minimum spanning networks, and the analysis of molecular variance showed the existence of a hierarchical structure. At the first level, two genetic groups were found, one containing ‘Reine Claude’ type reference cultivars altogether with ca. 25% of local genotypes, and a second one much more diverse. This latter group split in two groups, one containing most (ca. 70%) local genotypes and some old Spanish and French reference cultivars, whereas the other included 24 reference cultivars and only six local genotypes. A third partition level allowed a significant finer delineation into five groups. As a whole, the genetic structure of European plum from Northeastern Spain was shown to be complex and conditioned by a geographical proximity factor. This study not only contributes to genetic conservation and breeding for this species at the national level, but also supports the relevance of undertaking similar tasks of collection and characterization in other unexplored areas. Moreover, this kind of research could lead to future coordinated actions for the examination of the whole European plum diversity, to define conservation strategies, and could be used to better understand the genetic control of traits of horticultural interest through association mapping.

Development of a standardized methodology for phenotypical characterizations in apple

4 Pags.- 2 Tabls. Articles derived from XIV EUCARPIA Symposium on Fruit Breeding and Genetics (Bologna,Italy. June 14-18 2015) . The definitive version is available at: http://www.actahort.org/index.htm

Additional file 3: of Analysis of the genetic diversity and structure across a wide range of germplasm reveals prominent gene flow in apple at the European level

Characteristics of the 16 SSR markers used in this study with indication of the corresponding multiplex and dye. Footnotes: a [61]; b [60]; c [59]; d [62]; e Primer concentration within a given multiplex has been adjusted to get more homogeneous SSR marker amplification intensities. (XLSX 10 kb)