Identification of novel genetic regions associated with resistance to European canker in apple.

BACKGROUND: European canker, caused by the fungal pathogen Neonectria ditissima, is an economically damaging disease in apple producing regions of the world - especially in areas with moderate temperatures and high rainfall. The pathogen has a wide host range of hardwood perennial species, causing trunk cankers, dieback and branch lesions in its hosts. Although apple scion germplasm carrying partial resistance to the disease has been described, little is still known of the genetic basis for this quantitative resistance. RESULTS: Resistance to Neonectria ditissima was studied in a multiparental population of apple scions using several phenotyping methods. The studied population consists of individuals from multiple families connected through a common pedigree. The degree of disease of each individual in the population was assessed in three experiments: artificial inoculations of detached dormant shoots, potted trees in a glasshouse and in a replicated field experiment. The genetic basis of the differences in disease was studied using a pedigree-based analysis (PBA). Three quantitative trait loci (QTL), on linkage groups (LG) 6, 8 and 10 were identified in more than one of the phenotyping strategies. An additional four QTL, on LG 2, 5, 15 and 16 were only identified in the field experiment. The QTL on LG2 and 16 were further validated in a biparental population. QTL effect sizes were small to moderate with 4.3 to 19% of variance explained by a single QTL. A subsequent analysis of QTL haplotypes revealed a dynamic response to this disease, in which the estimated effect of a haplotype varied over the field time-points. CONCLUSIONS: This study describes the first identified QTL associated with resistance to N. ditissima in apple scion germplasm. The results from this study show that QTL present in germplasm commonly used in apple breeding have a low to medium effect on resistance to N. ditissima. Hence, multiple QTL will need to be considered to improve resistance through breeding.

SSR-Based Analysis of Genetic Diversity and Structure of Sweet Cherry (Prunus avium L.) from 19 Countries in Europe.

Sweet cherry (Prunus avium L.) is a temperate fruit species whose production might be highly impacted by climate change in the near future. Diversity of plant material could be an option to mitigate these climate risks by enabling producers to have new cultivars well adapted to new environmental conditions. In this study, subsets of sweet cherry collections of 19 European countries were genotyped using 14 SSR. The objectives of this study were (i) to assess genetic diversity parameters, (ii) to estimate the levels of population structure, and (iii) to identify germplasm redundancies. A total of 314 accessions, including landraces, early selections, and modern cultivars, were monitored, and 220 unique SSR genotypes were identified. All 14 loci were confirmed to be polymorphic, and a total of 137 alleles were detected with a mean of 9.8 alleles per locus. The average number of alleles (N = 9.8), PIC value (0.658), observed heterozygosity (Ho = 0.71), and expected heterozygosity (He = 0.70) were higher in this study compared to values reported so far. Four ancestral populations were detected using STRUCTURE software and confirmed by Principal Coordinate Analysis (PCoA), and two of them (K1 and K4) could be attributed to the geographical origin of the accessions. A N-J tree grouped the 220 sweet cherry accessions within three main clusters and six subgroups. Accessions belonging to the four STRUCTURE populations roughly clustered together. Clustering confirmed known genealogical data for several accessions. The large genetic diversity of the collection was demonstrated, in particular within the landrace pool, justifying the efforts made over decades for their conservation. New sources of diversity will allow producers to face challenges, such as climate change and the need to develop more sustainable production systems.

Towards a Joint International Database: Alignment of SSR Marker Data for European Collections of Cherry Germplasm.

The objective of our study was the alignment of microsatellite or simple sequence repeat (SSR) marker data across germplasm collections of cherry within Europe. Through the European Cooperative program for Plant Genetic Resources ECPGR, a number of European germplasm collections had previously been analysed using standard sets of SSR loci. However, until now these datasets remained unaligned. We used a combination of standard reference genotypes and ad-hoc selections to compile a central dataset representing as many alleles as possible from national datasets produced in France, Great Britain, Germany, Italy, Sweden and Switzerland. Through the comparison of alleles called in data from replicated samples we were able to create a series of alignment factors, supported across 448 different allele calls, that allowed us to align a dataset of 2241 SSR profiles from six countries. The proportion of allele comparisons that were either in agreement with the alignment factor or confounded by null alleles ranged from 67% to 100% and this was further improved by the inclusion of a series of allele-specific adjustments. The aligned dataset allowed us to identify groups of previously unknown matching accessions and to identify and resolve a number of errors in the prior datasets. The combined and aligned dataset represents a significant step forward in the co-ordinated management of field collections of cherry in Europe.

The apple REFPOP-a reference population for genomics-assisted breeding in apple.

Breeding of apple is a long-term and costly process due to the time and space requirements for screening selection candidates. Genomics-assisted breeding utilizes genomic and phenotypic information to increase the selection efficiency in breeding programs, and measurements of phenotypes in different environments can facilitate the application of the approach under various climatic conditions. Here we present an apple reference population: the apple REFPOP, a large collection formed of 534 genotypes planted in six European countries, as a unique tool to accelerate apple breeding. The population consisted of 269 accessions and 265 progeny from 27 parental combinations, representing the diversity in cultivated apple and current European breeding material, respectively. A high-density genome-wide dataset of 303,239 SNPs was produced as a combined output of two SNP arrays of different densities using marker imputation with an imputation accuracy of 0.95. Based on the genotypic data, linkage disequilibrium was low and population structure was weak. Two well-studied phenological traits of horticultural importance were measured. We found marker-trait associations in several previously identified genomic regions and maximum predictive abilities of 0.57 and 0.75 for floral emergence and harvest date, respectively. With decreasing SNP density, the detection of significant marker-trait associations varied depending on trait architecture. Regardless of the trait, 10,000 SNPs sufficed to maximize genomic prediction ability. We confirm the suitability of the apple REFPOP design for genomics-assisted breeding, especially for breeding programs using related germplasm, and emphasize the advantages of a coordinated and multinational effort for customizing apple breeding methods in the genomics era.

Development of a minimal KASP marker panel for distinguishing genotypes in apple collections.

Accurate identification of named accessions in germplasm collections is extremely important, especially for vegetatively propagated crops which are expensive to maintain. Thus, an inexpensive, reliable, and rapid genotyping method is essential because it avoids the need for laborious and time-consuming morphological comparisons. Single Nucleotide Polymorphism (SNP) marker panels containing large numbers of SNPs have been developed for many crop species, but such panels are much too large for basic cultivar identification. Here, we have identified a minimum set of SNP markers sufficient to distinguish apple cultivars held in the English and Welsh national collections providing a cheaper and automatable alternative to the markers currently used by the community. We show that SNP genotyping with a small set of well selected markers is equally efficient as microsatellites for the identification of apple cultivars and has the added advantage of automation and reduced cost when screening large numbers of samples.

Using whole-genome SNP data to reconstruct a large multi-generation pedigree in apple germplasm.

BACKGROUND: Apple (Malus x domestica Borkh.) is one of the most important fruit tree crops of temperate areas, with great economic and cultural value. Apple cultivars can be maintained for centuries in plant collections through grafting, and some are thought to date as far back as Roman times. Molecular markers provide a means to reconstruct pedigrees and thus shed light on the recent history of migration and trade of biological materials. The objective of the present study was to identify relationships within a set of over 1400 mostly old apple cultivars using whole-genome SNP data (~ 253 K SNPs) in order to reconstruct pedigrees. RESULTS: Using simple exclusion tests, based on counting the number of Mendelian errors, more than one thousand parent-offspring relations and 295 complete parent-offspring families were identified. Additionally, a grandparent couple was identified for the missing parental side of 26 parent-offspring pairings. Among the 407 parent-offspring relations without a second identified parent, 327 could be oriented because one of the individuals was an offspring in a complete family or by using historical data on parentage or date of recording. Parents of emblematic cultivars such as 'Ribston Pippin', 'White Transparent' and 'Braeburn' were identified. The overall pedigree combining all the identified relationships encompassed seven generations and revealed a major impact of two Renaissance cultivars of French and English origin, namely 'Reinette Franche' and 'Margil', and one North-Eastern Europe cultivar from the 1700s, 'Alexander'. On the contrary, several older cultivars, from the Middle Ages or the Roman times, had no, or only single, identifiable offspring in the set of studied accessions. Frequent crosses between cultivars originating from different European regions were identified, especially from the nineteenth century onwards. CONCLUSIONS: The availability of over 1400 apple genotypes, previously filtered for genetic uniqueness and providing a broad representation of European germplasm, has been instrumental for the success of this large pedigree reconstruction. It enlightens the history of empirical selection and recent breeding of apple cultivars in Europe and provides insights to speed-up future breeding and selection.

Genetic analysis of a major international collection of cultivated apple varieties reveals previously unknown historic heteroploid and inbred relationships.

Domesticated apple (Malus x domestica Borkh.) is a major global crop and the genetic diversity held within the pool of cultivated varieties is important for the development of future cultivars. The aim of this study was to investigate the diversity held within the domesticated form, through the analysis of a major international germplasm collection of cultivated varieties, the UK National Fruit Collection, consisting of over 2,000 selections of named cultivars and seedling varieties. We utilised Diversity Array Technology (DArT) markers to assess the genetic diversity within the collection. Clustering attempts, using the software STRUCTURE revealed that the accessions formed a complex and historically admixed group for which clear clustering was challenging. Comparison of accessions using the Jaccard similarity coefficient allowed us to identify clonal and duplicate material as well as revealing pairs and groups that appeared more closely related than a standard parent-offspring or full-sibling relations. From further investigation, we were able to propose a number of new pedigrees, which revealed that some historically important cultivars were more closely related than previously documented and that some of them were partially inbred. We were also able to elucidate a number of parent-offspring relationships that had resulted in a number of important polyploid cultivars. This included reuniting polyploid cultivars that in some cases dated as far back as the 18th century, with diploid parents that potentially date back as far as the 13th century.

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.

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

BACKGROUND: The 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 (North + East, West and South) with the aim to analyze the extent, distribution and structure of variation in the apple genetic resources in Europe. RESULTS: A Bayesian model-based clustering approach showed that diversity was organized in three groups, although these were only moderately differentiated (FST = 0.031). 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 (FST = 0.044). 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. CONCLUSIONS: The 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.

Development of colour and firmness in strawberry crops is UV light sensitive, but colour is not a good predictor of several quality parameters.

BACKGROUND: Strawberry (Fragaria × ananassa Duchesne var. Elsanta) plants were grown in polytunnels covered with three polythene films that transmitted varying levels of ultraviolet (UV) light. Fruit were harvested under near-commercial conditions and quality and yield were measured. During ripening, changes in the colour parameters of individual fruit were monitored, and the accuracy of using surface colour to predict other quality parameters was determined by analysing the correlation between colour and quality parameters within UV treatments. RESULTS: Higher exposure to UV during growth resulted in the fruit becoming darker at harvest and developing surface colour more quickly; fruit were also firmer at harvest, but shelf life was not consistently affected by the UV regime. Surface colour measurements were poorly correlated to firmness, shelf life or total phenolics, anthocyanins and ellagic acid contents. CONCLUSION: Although surface colour of strawberry fruits was affected by the UV regime during growth, and this parameter is an important factor in consumer perception, we concluded that the surface colour at the time of harvest was, contrary to consumer expectations, a poor indicator of firmness, potential shelf life or anthocyanin content.

Changes in the flavonoid and phenolic acid contents and antioxidant activity of red leaf lettuce (Lollo Rosso) due to cultivation under plastic films varying in ultraviolet transparency.

Red leaf lettuce (Lollo Rosso) was grown under three types of plastic films that varied in transparency to UV radiation (designated as UV block, UV low, and UV window). Flavonoid composition was determined by high-performance liquid chromatography (HPLC), total phenolics by the Folin-Ciocalteu assay, and antioxidant capacity by the oxygen radical absorbance capacity (ORAC) assay. Exposure to increased levels of UV radiation during cultivation caused the leaves to redden and increased concentrations of total phenols and the main flavonoids, quercetin and cyanidin glycosides, as well as luteolin conjugates and phenolic acids. The total phenol content increased from 1.6 mg of gallic acid equivalents (GAE)/g of fresh weight (FW) for lettuce grown under UV block film to 2.9 and 3.5 mg of GAE/g of FW for lettuce grown under the UV low and UV window films. The antioxidant activity was also higher in lettuce exposed to higher levels of UV radiation with ORAC values of 25.4 and 55.1 micromol of Trolox equivalents/g of FW for lettuce grown under the UV block and UV window films, respectively. The content of phenolic acids, quantified as caffeic acid, was also different, ranging from 6.2 to 11.1 micromol/g of FW for lettuce cultivated under the lowest and highest UV exposure plastic films, respectively. Higher concentrations of the flavonoid glycosides were observed with increased exposure to UV radiation, as demonstrated by the concentrations of aglycones after hydrolysis, which were cyanidin (ranging from 165 to 793 microg/g), quercetin (ranging from 196 to 880 microg/g), and luteolin (ranging from 19 to 152 microg/g). The results demonstrate the potential of the use of UV-transparent plastic as a means of increasing beneficial flavonoid content of red leaf lettuce when the crop is grown in polytunnels.

Floral meristem indeterminacy depends on flower position and is facilitated by acarpellate gynoecium development in Impatiens balsamina.

* Floral meristems are generally determinate. Termination of their activity varies with species, occurring after carpel or ovule development, depending on the placentation type. In terminal flowering Impatiens balsamina (cv. Dwarf Bush Flowered) some flowers exhibit meristem indeterminacy; they produce organs from the placenta after ovule development. * Here we provide a detailed description of gynoecium development in this line and explore the basis of the indeterminate nature of some of its floral meristems. * We find that the placenta is sometimes established without complete carpel fusion. Proliferative growth derives from meristematic remnants of the placenta and is more common in the terminal inflorescence. RNA in situ hybridization reveals that IbLFY (Impatiens LFY homologue) is expressed in all meristem states, even in proliferating meristems. Expression of IbAG in axillary flowers is as expected in the meristem, stamens and carpels but absent from the proliferating meristem. * We conclude that I. balsamina has cauline placentation. Incomplete suppression of inflorescence identity in flowers of the terminal inflorescence leads to floral meristem proliferation after ovule development in this species.

LEAFY, TERMINAL FLOWER1 and AGAMOUS are functionally conserved but do not regulate terminal flowering and floral determinacy in Impatiens balsamina.

In Impatiens balsamina a lack of commitment of the meristem during floral development leads to the continuous requirement for a leaf-derived floral signal. In the absence of this signal the meristem reverts to leaf production. Current models for Arabidopsis state that LEAFY (LFY) is central to the integration of floral signals and regulates flowering partly via interactions with TERMINAL FLOWER1 (TFL1) and AGAMOUS (AG). Here we describe Impatiens homologues of LFY, TFL1 and AG (IbLFY, IbTFL1 and IbAG) that are highly conserved at a sequence level and demonstrate homologous functions when expressed ectopically in transgenic Arabidopsis. We relate the expression patterns of IbTFL1 and IbAG to the control of terminal flowering and floral determinacy in Impatiens. IbTFL1 is involved in controlling the phase of the axillary meristems and is expressed in axillary shoots and axillary meristems which produce inflorescences, but not in axillary flowers. It is not involved in maintaining the terminal meristem in either an inflorescence or indeterminate state. Terminal flowering in Impatiens appears therefore to be controlled by a pathway that uses a different integration system than that regulating the development of axillary flowers and branches. The pattern of ovule production in Impatiens requires the meristem to be maintained after the production of carpels. Consistent with this morphological feature IbAG appears to specify stamen and carpel identity, but is not sufficient to specify meristem determinacy in Impatiens.

Mechanisms and function of flower and inflorescence reversion.

Flower and inflorescence reversion involve a switch from floral development back to vegetative development, thus rendering flowering a phase in an ongoing growth pattern rather than a terminal act of the meristem. Although it can be considered an unusual event, reversion raises questions about the nature and function of flowering. It is linked to environmental conditions and is most often a response to conditions opposite to those that induce flowering. Research on molecular genetic mechanisms underlying plant development over the last 15 years has pinpointed some of the key genes involved in the transition to flowering and flower development. Such investigations have also uncovered mutations which reduce floral maintenance or alter the balance between vegetative and floral features of the plant. How this information contributes to an understanding of floral reversion is assessed here. One issue that arises is whether floral commitment (defined as the ability to continue flowering when inductive conditions no longer exist) is a developmental switch affecting the whole plant or is a mechanism which assigns autonomy to individual meristems. A related question is whether floral or vegetative development is the underlying default pathway of the plant. This review begins by considering how studies of flowering in Arabidopsis thaliana have aided understanding of mechanisms of floral maintenance. Arabidopsis has not been found to revert to leaf production in any of the conditions or genetic backgrounds analysed to date. A clear-cut reversion to leaf production has, however, been described in Impatiens balsamina. It is proposed that a single gene controls whether Impatiens reverts or can maintain flowering when inductive conditions are removed, and it is inferred that this gene functions to control the synthesis or transport of a leaf-generated signal. But it is also argued that the susceptibility of Impatiens to reversion is a consequence of the meristem-based mechanisms controlling development of the flower in this species. Thus, in Impatiens, a leaf-derived signal is critical for completion of flowering and can be considered to be the basis of a plant-wide floral commitment that is achieved without accompanying meristem autonomy. The evidence, derived from in vitro and other studies, that similar mechanisms operate in other species is assessed. It is concluded that most species (including Arabidopsis) are less prone to reversion because signals from the leaf are less ephemeral, and the pathways driving flower development have a high level of redundancy that generates meristem autonomy even when leaf-derived signals are weak. This gives stability to the flowering process, even where its initiation is dependent on environmental cues. On this interpretation, Impatiens reversion appears as an anomaly resulting from an unusual combination of leaf signalling and meristem regulation. Nevertheless, it is shown that the ability to revert can serve a function in the life history strategy (perenniality) or reproductive habit (pseudovivipary) of many plants. In these instances reversion has been assimilated into regular plant development and plays a crucial role there.