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Breeding fruit species is time-consuming and expensive. With few exceptions, trees are likely the worst species to work with in terms of genetics and breeding. Most are characterized by large trees, long juvenile periods, and intensive agricultural practice, and environmental variability plays an important role in the heritability evaluations of every single important trait. Although vegetative propagation allows for the production of a significant number of clonal replicates for the evaluation of environmental effects and genotype × environment interactions, the spaces required for plant cultivation and the intensity of work necessary for phenotypic surveys slow down the work of researchers. Fruit breeders are very often interested in fruit traits: size, weight, sugar and acid content, ripening time, fruit storability, and post-harvest practices, among other traits relevant to each individual species. The translation of trait loci and whole-genome sequences into diagnostic genetic markers that are effective and affordable for use by breeders, who must choose genetically superior parents and subsequently choose genetically superior individuals among their progeny, is one of the most difficult tasks still facing tree fruit geneticists. The availability of updated sequencing techniques and powerful software tools offered the opportunity to mine tens of fruit genomes to find out sequence variants potentially useful as molecular markers. This review is devoted to analysing what has been the role of molecular markers in assisting breeders in selection processes, with an emphasis on the fruit traits of the most important fruit crops for which examples of trustworthy molecular markers have been developed, such as the MDo.chr9.4 marker for red skin colour in apples, the CCD4-based marker CPRFC1, and LG3_13.146 marker for flesh colour in peaches, papayas, and cherries, respectively.

Dioecism and an extended juvenile phase of 3–7 years in kiwifruit hinder the progress in breeding new cultivars. The identification of fruit-bearing females at an early stage of growth is crucial for breeders. Consequently, molecular markers have become a key tool for identifying female and male plants at an early stage of development. Several efforts were made to identify PCR-based sex linked markers in Actinidia; however, those markers are characterized by a highly polymorphic nature affecting the result of the screening reliability, suggesting the need of more suitable, stable markers, characterized by a consistent transferability among genotypes and species. The main goal of this work was to develop a method for the ultimate discrimination of females from male plants at an early stage of growth using sex-linked markers. We developed an Early Sex Discrimination molecular Test (ESD Test) that allows the discrimination of male and female plants using a simple PCR amplification test. We demonstrate that the test could unequivocally identify the gender of an unknown sample both in the most commercially important species A. chinensis and in further 13 Actinidia species tested with the exception of Actinidia latifolia, where markers fail in gender discrimination. Male genotypes could be easily identified and discarded reducing the cost of a breeding program.

This paper reports the genetic diversity of apple germplasm collected in the Friuli Venezia Giulia region (north-eastern Italy). The collection, maintained in three different locations, was represented by local cultivars which probably originated as chance seedlings together with cultivars introduced from neighbouring countries, the name and origin of most of which has been lost over time. A preliminary procedure described in the paper started with 469 molecular profiles analysed using 15 Simple Sequence Repeat (SSR) markers and allowed identification of the ‘true-to-type’ genotypes among those maintained in multiple locations. The set of the remaining 234 accessions was further reduced to 132 unique profiles by removing 102 synonyms, that is accessions with different name and the same molecular profile. Flow cytometry identified as many as 54 triploids (40.9%), whose status was confirmed by field observations on leaf size and the occurrence of triallelic profiles at several SSR markers. The remaining 78 diploid accessions were analysed for their genetic diversity, that is the number of alleles, observed and expected heterozygosity, polymorphism information content (PIC), the frequency of null alleles and the probability of identity for unrelated and full-sib genotypes. The paper provides a critical evaluation of the SSR markers adopted for the study, discusses the genetic diversity observed in the apple germplasm collection examined as well as its high frequency of triploids compared with other apple collections described in the literature.

Waxy apple cuticles predominantly accumulate ursane-type triterpenes, but the profile shifts with the induction of skin russeting towards lupane-type triterpenes. We previously characterised several key enzymes in the ursane-type and lupane-type triterpene pathways, but this switch in triterpene metabolism associated with loss of cuticle integrity is not fully understood. To analyse the relationship between triterpene biosynthesis and russeting, we used microscopy, RNA-sequencing and metabolite profiling during apple fruit development. We compared the skin of three genetically-close clones of ‘Golden Delicious’ (with waxy, partially russeted and fully russeted skin). We identified a unique molecular profile for the russet clone, including low transcript abundance of multiple cuticle-specific metabolic pathways in the early stages of fruit development. Using correlation analyses between gene transcription and metabolite concentration we found MYB transcription factors strongly associated with lupane-type triterpene biosynthesis. We showed how their transcription changed with the onset of cuticle cracking followed by russeting and that one factor, MYB66, was able to bind the promoter of the oxidosqualene cyclase OSC5, to drive the production of lupeol derivatives. These results provide insights into the breakdown of cuticle integrity leading to russet and how this drives MYB-regulated changes to triterpene biosynthesis.

International audience; Apple scab, caused by Venturia inaequalis, is a major disease worldwide. To control this disease, up to 20-25 fungicide applications may be needed depending on the year and the production system. Cultivation of scab-resistant apple cultivars would reduce the chemical footprint of apple production. Breeding for durable scab resistance is an objective of most apple breeding programmes. One way to achieve durable resistance is to pyramid multiple apple scab resistance genes in a cultivar. Currently, more than 18 different apple scab resistance genes have been reported. Molecular markers are available for most of these and can be used to select seedlings carrying multiple resistance genes. Apple scab isolates are virulent to specific apple scab resistance genes. Choice of optimal resistance genes for use in a breeding program, should therefore take regional presence of these apple scab isolates into consideration. In order to learn more about the geographical distribution of apple scab isolates, the VINQUEST project (www.vinquest.ch) was initiated in 2009. Ten years after the launch of this initiative, 24 partners from 14 countries are regularly contributing data. Results obtained from the data collected during the first 10 years provide the first insights into which apple scab resistance genes may hold most promise for breeding cultivars with durable resistance.

BackgroundVitis viniferaL. is the most cultivated grapevine species worldwide.Erysiphe necatorSch., the causal agent of grape powdery mildew, is one of the main pathogens affecting viticulture.V. viniferahas little or no genetic resistances againstE. necatorand the grape industry is highly dependent on agrochemicals. Some CaucasianV. viniferaaccessions have been reported to be resistant toE. necatorand to have no genetic relationships to known sources of resistance to powdery mildew. The main purpose of this work was the study and mapping of the resistance toE. necatorin the Caucasian grapes ‘Shavtsitska’ and ‘Tskhvedianis tetra’.ResultsThe Caucasian varieties ‘Shavtsitska’ and ‘Tskhvedianis tetra’ showed a strong partial resistance toE. necatorwhich segregated in two cross populations: the resistant genotypes delayed and limited the pathogen mycelium growth, sporulation intensity and number of conidia generated. A total of 184 seedlings of ‘Shavtsitska’ x ‘Glera’ population were genotyped through the Genotyping by Sequencing (GBS) technology and two high-density linkage maps were developed for the cross parents. The QTL analysis revealed a major resistance locus, explaining up to 80.15% of the phenotypic variance, on ‘Shavtsitska’ linkage group 13, which was associated with a reduced pathogen infection as well as an enhanced plant necrotic response. The genotyping of 105 Caucasian accessions with SSR markers flanking the QTL revealed that the resistant haplotype of ‘Shavtsitska’ was shared by ‘Tskhvedianis tetra’ and a total of 25 Caucasian grape varieties, suggesting a widespread presence of this resistance in the surveyed germplasm. The uncovered QTL was mapped in the region where theRen1locus of resistance toE. necator, identified in theV. vinifera‘Kishmish vatkana’ and related grapes of Central Asia, is located. The genetic analysis conducted revealed that the Caucasian grapes in this study exhibit a resistant haplotype different from that of Central Asian grape accessions.ConclusionsThe QTL isolated in ‘Shavtsitska’ and present in the CaucasianV. viniferavarieties could be a new candidate gene of resistance toE. necatorto use in breeding programmes. It co-localizes with theRen1locus but shows a different haplotype from that of grapevines of Central Asia. We therefore consider that the Caucasian resistance locus, namedRen1.2, contains a member of a cluster of R-genes, of which the region is rich, and to be linked with, or possibly allelic, toRen1.

International audience; Apple scab, caused by Venturia inaequalis, is a major fungal disease worldwide. Cultivation of scab-resistant cultivars would reduce the chemical footprint of apple production. However, new apple cultivars carrying durable resistances should be developed to prevent or at least slow the breakdown of resistance against races of V. inaequalis. One way to achieve durable resistance is to pyramid multiple scab resistance genes in a cultivar. The choice of the resistance genes to be combined in the pyramids should take into account the frequency of resistance breakdown and the geographical distribution of apple scab isolates able to cause such breakdowns. In order to acquire this information and to make it available to apple breeders, the VINQUEST project (www.vinquest.ch) was initiated in 2009. Ten years after launching this project, 24 partners from 14 countries regularly contribute data. From 2009 to 2018 nearly 9000 data points have been collected. This information has been used to identify the most promising apple scab resistance genes for developing cultivars with durable resistance, which to date are: Rvi5, Rvi11, Rvi12, Rvi14 and Rvi15. As expected, Rvi1, together with Rvi3 and Rvi8, were often overcome, hence have little value for scab resistance breeding. Rvi10 may also belong to this group. On the other hand Rvi2, Rvi4, Rvi6, Rvi7, Rvi9, and Rvi13 are still useful for the breeding, but their use is recommended only in extended pyramids of (≥3) resistance genes.

In the present study, eight early-ripening apricot cultivars of the Italian and international germplasm were evaluated during three years of investigation (2013–2015), according to their quality traits, biochemical composition, antiradical capacity and hydroxycinnamic acid (HCA) profile, using a chemometric approach. Among cultivars analysed, the highest fresh weight was detected in ‘Maia’ (106 ± 3 g), whereas the lightest fruits were produced by ‘Ottavianese’ cv (51 ± 4 g). ‘Orange Rubis’ and ‘Maia’ were characterized by the highest levels of dry matter, which represent a key processing parameter for the production of dried apricots. The highest content of bioactive compounds such as total phenols and flavans were found in ‘Spring Blush’, Orange Rubis’ and ‘Monaco Bello’, indicating a greater susceptibility to browning during processing. Chlorogenic acid was the most abundant hydroxycinnamic acid found in all tested genotypes, being ‘Portici’ the richest one (338 ± 21 μg g −1 on dry basis). High significant correlations were found among parameters analysed, especially between colour coordinates and bioactive compounds. Finally, application of stepwise discriminant analysis to apricot samples showed that total phenols, total flavans and antiradical capacity were the most important variables to differentiate the cultivars analysed. Besides, principal component analysis made it possible to establish similarities among the cultivars depending on their quality and biochemical characteristics. It is concluded that the combination of quality, biochemical traits analysis and chemometric techniques can be used as a consistent procedure to provide breeders with useful information about the identification and characterization of the most promising early ripening genotypes, both for fresh consumption and processing.

A wild grape haplotype (Rpv3‐1) confers resistance to Plasmopara viticola. We mapped the causal factor for resistance to an interval containing a TIR‐NB‐LRR (TNL) gene pair that originated 1.6–2.6 million years ago by a tandem segmental duplication. Transient coexpression of the TNL pair in Vitis vinifera leaves activated pathogen‐induced necrosis and reduced sporulation compared with control leaves. Even though transcripts of the TNL pair from the wild haplotype appear to be partially subject to nonsense‐mediated mRNA decay, mature mRNA levels in a homozygous resistant genotype were individually higher than the mRNA trace levels observed for the orthologous single‐copy TNL in sensitive genotypes. Allelic expression imbalance in a resistant heterozygote confirmed that cis‐acting regulatory variation promotes expression in the wild haplotype. The movement of transposable elements had a major impact on the generation of haplotype diversity, altering the DNA context around similar TNL coding sequences and the GC‐content in their proximal 5′‐intergenic regions. The wild and domesticated haplotypes also diverged in conserved single‐copy intergenic DNA, but the highest divergence was observed in intraspecific and not in interspecific comparisons. In this case, introgression breeding did not transgress the genetic boundaries of the domesticated species, because haplotypes present in modern varieties sometimes predate speciation events between wild and cultivated species.

Apple breeding is active worldwide and yet the apple crop is in a precarious state as it relies on few dominant cultivars and only the Rvi6 (formerly Vf) gene, that confers resistance to scab, has been extensively exploited in the cultivars entered the market in recent years. However, there are some 20 disease resistance genes described in apple and the apple germplasm includes thousands of accessions in the repositories. In this paper, a breeding programme is described, whereby 36 genotypes, including ancient and contemporary apple cultivars, were crossed to produce a new set of selections that combine extensive genetic resources with pyramided resistance genes to several apple diseases, such as scab and powdery mildew. The 110 cross combinations carried out successfully, of the 260 initially planned, produced 7,876 offsprings, reduced to 2,969 after screening with molecular markers associated with five resistance genes. Selections with three or two resistance genes and good agronomic characteristics were kept for further field observations with the aims of creating new cultivars for the market and new parents for future breeding projects

In this chapter we present a review on the studies and analyses of grapevine genome, from the progress on markers to identify the segregating alleles in the crosses between varieties, to the assembly of genome sequences and to transcriptomic studies. The focus of this chapter is on resistance genes, either dominant Nucleotide binding-Leucine Rich Repeat Receptors (NLRs), or the non-host resistance genes, such as proteins regulating intracellular trafficking, and transcription factors. Lastly, a role of secondary metabolites in response to pathogens is discussed. A second line of defense is presented, based on small RNAs and RNA silencing, that is targeted by pathogen Silencing Suppressors. Finally, the potential future developments based on nanoparticles, extracellular vesicles, nanobodies, cross-kingdom RNA trafficking, and delivery of naked or enveloped nucleic acids is discussed.

The major goal of landscape genetics is to understand how landscape structure genetic variation in natural populations. We investigated molecular diversity in Acer opalus subsp. obtusatum sampled from 95 sites using 14 nuclear microsatellite loci. The average number of alleles per nuclear microsatellite locus differed among sampling sites; the number was high (4.9 alleles) in populations from the Basilicata and Molise regions, where heterozygosity was also high (0.679, Molise; 0.669, Basilicata). Differentiation between sites was often low (mean FST = 0.220), indicating few genetic differences between most sites. There was a clear excess of homozygotes (mean Ho = 0.450, mean He = 0.513) and a relatively high FIS (mean = 0.451), suggesting a consistent level of inbreeding in many A. opalus subsp. obtusatum populations. There was a significant pattern of isolation by distance across the study area (Mantel test; R2 = 0.0662, P

Background Russeting is a disorder developed by apple fruits that consists of cuticle cracking followed by the replacement of the epidermis by a corky layer that protects the fruit surface from water loss and pathogens. Although influenced by many environmental conditions and orchard management practices, russeting is under genetic control. The difficulty in classifying offspring and consequent variable segregation ratios have led several authors to conclude that more than one genetic determinant could be involved, although some evidence favours a major gene (Ru). Results In this study we report the mapping of a major genetic russeting determinant on linkage group 12 of apple as inferred from the phenotypic observation in a segregating progeny derived from ‘Renetta Grigia di Torriana’, the construction of a 20 K Illumina SNP chip based genetic map, and QTL analysis. Recombination analysis in two mapping populations restricted the region of interest to approximately 400 Kb. Of the 58 genes predicted from the Golden Delicious sequence, a putative ABCG family transporter has been identified. Within a small set of russeted cultivars tested with markers of the region, only six showed the same haplotype of ‘Renetta Grigia di Torriana’. Conclusions A major determinant (Ru_RGT) for russeting development putatively involved in cuticle organization is proposed as a candidate for controlling the trait. SNP and SSR markers tightly co-segregating with the Ru_RGT locus may assist the breeder selection. The observed segregations and the analysis of the ‘Renetta Grigia di Torriana’ haplotypic region in a panel of russeted and non-russeted cultivars may suggest the presence of other determinants for russeting in apple. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0507-4) contains supplementary material, which is available to authorized users.

Juglans regia L. is distributed primarily across temperate and subtropical regions of the Northern Hemisphere. During the last glaciation, the species survived in refugial areas that in Europe included the Balkans and the Italian peninsula, two areas joined by a corridor represented by the Friuli Venezia Giulia region, where two germplasm reservoirs met and likely intercrossed during re-colonization after the last glaciation. In this work, two hundred and fifteen wild accessions native to the area were sampled, georeferenced, and genotyped with 20 microsatellite loci selected from the literature. The local accessions of this study displayed moderate genetic diversity with 80 alleles identified. The number of alleles/loci was 4.0 (4.7 alleles for the genomic SSRs (Simple Sequence Repeats) and 2.7 alleles per EST (Expressed Sequence Tag)-derived SSR, on average). An analysis of molecular variance (AMOVA) revealed that most of the molecular diversity was between individuals (nearly 98% of variation explained). The model-based clustering algorithms implemented either in STRUCTURE and GENELAND software revealed two clusters: The first one encompassed most of the samples and showed a great genetic admixture throughout the five sampling areas defined on the base of orographic characteristics of the region. The second cluster represented a small island with three samples traced back to an introduction from Russia at the beginning of the 20th century.

Nuts collected from wild accessions of Persian walnut (Juglans regia L.) in the Friuli Venezia Giulia region (North Eastern Italian Alps) were evaluated during 2013–2015. The analyses carried out were mainly on fruit traits, the only morphological traits that could be observed and statistically analysed, being less dependent on the area of sampling, considering geographic features such as altitude, slope, soil, and climate. Such fruit traits proved to be very variable. The nut weight ranged from 2.2 to 17.3 g, the shell thickness from 0.35 to 2.30 mm, the color of kernel skin from light to amber. The fruit nut appearance, evaluated by a panel of consumers, varied from 2.50 to 6.83 in a scale from 0 (very bad) to 10 (very attractive). A multivariate analysis carried out considering traits valuable for breeding and selection, such as nut weight, kernel weight: nut weight ratio, shell thickness, kernel color, and fruit appearance, produced a ranking list, that included, at best, accessions that could be either selected as such for vegetative propagation and distribution to growers or used in breeding programs. A disadvantage of these selections is their terminal bearing habit which is not appreciated by breeders, who prefer the most productive genotypes with lateral bearing habit.

A collection of 60 accessions of Prunus avium L. from the sweet cherry germplasm repository of the Campania Region in Italy, together with seven reference accessions of P. avium, were screened to e...

The real origin (crossing partners) of rootstocks and their clones derived from Teleki’s seedlings are still unknown. Therefore, as a first step, we aimed at determining the genetic similarities or differences among these rootstocks by molecular tools. We have analysed 38 different rootstocks at seven microsatellite loci (VVMD5, VVMD6, VVMD7, VVMD28, VVMD31, VVS2, and VVS29). The genetic relatedness among the analysed clones can be inferred from the results presented in this paper.

The PCR–SSR technique was used to detect nuclear DNA diversity in five wild populations of Prunus avium from deciduous forests in Italy, Slovenia, and Croatia and 87 sweet cherry accessions from different geographical areas that have been maintained in the sweet cherry collection in Italy. This sweet cherry collection includes local accessions from the Campania Region as well as accessions from different countries. Twenty-eight microsatellites, previously developed in this species, generated polymorphic amplification products. Between 2 and 14 alleles were revealed for the polymorphic loci studied, with the expected heterozygosity ranging from 0.045 to 0.831. The total probability of identity was 56.94 × 10−18. A model-based Bayesian clustering analysis identified nine distinct gene pools in cultivated P. avium. The probability that wild populations were assigned to cultivated gene pools indicated that three gene pools accounted for the genomic origin of 53% of P. avium sampled. A dendrogram was generated using UPGMA (unweighted pair group method with arithmetic averages) based on Nei genetic distance analysis. This dendrogram classified most of the genotypes into one major group with an additional group of five accessions. The results indicate that this set of SSRs is highly informative, and they are discussed in terms of the implications for sweet cherry characterization.

Natural populations of species in the Acer genus occurring in Campania (southern Italy) were surveyed by screening seven microsatellite loci. Primer pairs for Acer pseudoplatanus L. microsatellite loci were analysed in six different species: Acer lobelii Ten., Acer campestre L., Acer pseudoplatanus L., Acer obtusatum W. et K., Acer neapolitanum Ten. and Acer monspessulanum L. The aim of the present study was to survey the genetic variability and genetic structure of natural populations of the Acer genus in Campania. The high degree of polymorphism observed in six different species of Acer makes these markers useful for investigating genetic variation at various spatial scales, and for the analysis of gene flow and of the mating system.

[EN]: We have identified a set of plants (the bin set) to permit “selective” or “bin” mapping using the diploid strawberry mapping population FV × FN, derived from the F2 cross F. vesca 815 × F. nubicola 601, which has been used to develop the Fragaria reference map. The bin set consists of 8 plants: the F. vesca 815 parent, the F1 hybrid individual, and 6 seedlings of the F2 population. This bin set divides the 578 cM of the diploid Fragaria genome into 46 bins, the largest mapping bin being 26 cM in length and the average bin size being 12.6 cM. To validate the FV × FN bin set, we used it to locate 103 loci into bins on the FV × FN map. These loci comprised 61 previously described SSRs, 38 new SSRs developed in this investigation from Fragaria × ananassa genomic DNA, EST and gene sequences, and 4 ripening-related genes developed for Prunus. The 103 markers were located to bins on all 7 linkage groups of the Fragaria map and a new mapping bin was identified with the novel markers, demonstrating that the map covers the majority of the diploid Fragaria genome and that the 6 bin-set seedlings selected were appropriate for bin mapping using this progeny., [FR]: Les auteurs ont identifié une collection d’individus (« bin set ») permettant une cartographie sélective (« bin mapping ») chez le fraisier diploïde à l’aide de la population de cartographie FV × FN. Ces individus sont tirés d’une population F2 issue du croisement F. vesca 815 × F. nubicola 601, laquelle a servi à produire la carte de référence pour le genre Fragaria. Cette collection comprend huit plantes : la parent F. vesca 815, l’individu F1 et six individus F2. Cette collection divise les 578 cM du génome du Fragaria diploïde en 46 segments (« bins ») dont la taille moyenne est de 12,6 cM et dont le plus grand mesure 26 cM. Afin de valider cette collection FV × FN, les auteurs l’ont employé pour assigner 103 locus à des segments de la carte FV × FN. Ces locus comprenaient 61 SSR développés antérieurement, 38 nouveaux SSR obtenus au cours de ce travail à partir de séquences génomiques, d’EST et de séquences géniques du Fragaria × ananassa, ainsi que quatre gènes impliqués dans le mûrissement chez le genre Prunus. Les 103 marqueurs ont été assignés à des segments sur les sept groupes de liaison de la carte du Fragaria et un nouveau segment a été identifié à l’aide des nouveaux marqueurs. Cela a permis de démontrer que la carte couvre la majeure partie du génome du Fragaria diploïde et que les six individus F2 de la collection sont adéquats pour la cartographie sélective au sein de cette population.

The development of molecular markers in kiwifruit followed the advances in molecular biology techniques. After occasional use of RFLP (restriction fragment length polymorphism) markers in the 1990s, most markers developed in kiwifruit were based on PCR (polymerase chain reaction). RAPD (random amplified polymorphic DNA) were followed by AFLPs (amplified fragment length polymorphisms), used mainly for the saturation of linkage maps. In the meantime, microsatellites or SSRs (simple sequence repeats) came to the fore and were extensively used for genotyping germplasm accessions and to produce linkage maps, where SSRs anchored markers for pairing parental maps of the pseudo-testcrosses and the identification of homologous linkage groups in maps produced from different cross populations. Four linkage maps for kiwifruit have been published: the first two based on SSR markers and the second two based mainly or exclusively on SNP markers identified and mapped at the same time through the approach of ‘genotyping-by-sequencing.’ These last, highly saturated, maps were produced to assist the genome-sequencing project, by anchoring and orientating scaffolds and contigs of the assembly. The linkage maps, in particular that published by the New Zealand Institute for Plant & Food Research based on 636 markers, mainly SSRs, helped in the mapping of several traits, such as gender that was mapped in the subtelomeric region of linkage group 17, corresponding to the pseudo-molecule number 25 of the genome assembly.

Kiwifruit breeding still largely relies on phenotypic observation of cross progeny grown in the field to fruiting maturity, without any selection prior to the juvenility being overcome. Developing markers for the selection of traits of interest would greatly help breeders to rapidly screen breeding populations. With the aim of mapping several traits of interest in kiwifruit, a F1 population of diploid (2n = 58) Actinidia chinensis was produced by combining parents with contrasting phenotypic traits. Ninety-four individuals were preliminarily analyzed to obtain a saturated genetic map based on 167 SSRs from the literature and 12,586 segregating restriction-site-associated DNA (RAD) loci obtained through an approach known as genotyping-by-sequencing (GBS) based on haplotype calling of SNP markers identified by a modified double digest restriction-associated DNA sequencing (ddRADseq) protocol as proposed by Peterson et al. (2012). To improve the accuracy of genotype calling, restriction-site-associated reads were aligned to the scaffolds of the recently published kiwifruit genome (Huang et al. 2013). This strategy provided genetic anchoring to 557 Mbp (90 %) of the assembly, helping also to anchor some 120 unmapped Mbp and to identify some mis-joined scaffolds. The analysis of the region controlling the dioecy in kiwifruit, spanning 16 scaffolds in the pseudomolecule 25 of the genome assembly (approximately 4.9 Mbp), with RAD markers that co-segregated with the gender determinant, allowed to sort out markers suitable for marker-assisted selection for the gender in the mapping population with successful extension to further controlled crosses having parents at different ploidy level and belonging to the A. chinensis/Actinidia deliciosa complex.

This study reports the development of 68 new microsatellite markers. Of these, 45 were obtained, together with 20 others already published, from an AC-enriched genomic library of the wild strawberry Fragaria vesca. The 68 markers were tested for transportability to the cultivated strawberry F. × ananassa 'Miss' and 83% gave positive amplifications. Twenty pairs of primers were selected and tested for their transportability to 16 Fragaria taxa and eight species of Rosaceae (peach, almond, apricot, European and Sino-Japanese plums, sweet and sour cherry, apple). The average proportion of primers amplifying loci in Fragaria was 69%, while the transportability to Rosaceae was very low and resulted in null amplification for 80% of the primer pairs. In addition, 23 microsatellite markers were developed from F. x ananassa 'expressed sequence tags' databases. A total of 141 primer pairs from these and published primers, were tested for polymorphism in the two parents (91.333.2 and 'Snovit'; both belonging to F. vesca) of a full sib population of 46 individuals. Fifty-eight percent of the primers were discarded because they were monomorphic, or were difficult to interpret, or their allelic conformation was not useful for mapping. The segregation of 73 primers was tested in the progeny and a partial map of the female parent was constructed, based on the segregation of 66 useful markers that were ordered into eight linkage groups of which four had from seven to 14 markers.

SummaryA collection of 48 accessions of Malus domestica Borkh. from the apple germplasm repository of the Campania Region in Italy, several of which were suspected to be “synonyms”, was screened together with eight ancient well-known cultivars, added for reference, using a set of nine microsatellite or Simple Sequence Repeat (SSR) primer pairs to determine genetic identities, to estimate genetic diversity and to identify genetic relationships between the accessions. All microsatellite (SSR) primers revealed polymorphism, with seven to 14 alleles per main locus and the expected heterozygosity (He) ranging from 0.713–0.884. Three SSRs (CH01h02, CH02c11 and CH04c06) amplified a second locus that was less polymorphic and clearly distinguished from the main locus. The frequency of each allele at the main loci was generally low, with many alleles detected in only one or a few cultivars. Of the 56 genotypes studied, as many as 27 were identified as “synonyms” and were excluded from the genetic analysis. Synonymy...

A kiwifruit (Actinidia deliciosa) natural sex mutant is described. The bud mutation occurred in a mature male vine and caused a gender change from male to female. The mutant (ser, sex reversal) carries apparently perfect flowers, with a well developed ovary and styles, but produces pollen that does not germinate and does not fertilise female genotypes. The ser mutant sets fruits when pollinated with pollen from the original or any other male, but not when selfed. The origin of the mutant as a sport mutation was confirmed by the identical genetic profile of both mutated and original canopy sectors at 12 microsatellite loci. Both the original and mutant parts of the vine were phenotypically stable on propagation by grafting. The paper discusses the likely genetic origin of the mutant and its usefulness for studying the genes that control dioecy in Actinidia.

A set of 109 microsatellite primer pairs recently developed for peach and cherry have been studied in the almond x peach F(2) progeny previously used to construct a saturated Prunus map containing mainly restriction fragment length polymorphism markers. All but one gave amplification products, and 87 (80%) segregated in the progeny and detected 96 loci. The resulting Prunus map contains a total of 342 markers covering a total distance of 522 cM. The approximate position of nine additional simple sequence repeats (SSRs) was established by comparison with other almond and peach maps. SSRs were placed in all the eight linkage groups of this map, and their distribution was relatively even, providing a genome-wide coverage with an average density of 5.4 cM/SSR. Twenty-four single-locus SSRs, highly polymorphic in peach, and each falling within 24 evenly spaced approximately 25-cM regions covering the whole Prunus genome, are proposed as a 'genotyping set' useful as a reference for fingerprinting, pedigree and genetic analysis of this species.

A genetic map of kiwifruit (Actinidia spp.) was constructed using microsatellite and AFLP markers and the pseudo-testcross mapping strategy. (AC)n and (AG)n microsatellite repeats were first isolated from Actinidia chinensis (2n = 2x = 58) enriched genomic libraries and tested for segregation in the interspecific cross between the diploid distantly related species A. chinensis and A. callosa. Some 105 microsatellite loci of the 251 initially tested segregated in the progeny in a 1:1 ratio as in a classical backcross, or in a ratio which could mimic the backcross, and were mapped using 94 individuals. AFLP markers were then produced using MseI and EcoRI restriction enzymes and 15 primer combinations. Nearly 10% of loci showed a distorted segregation at α = 0.05, and only 4% at α = 0.01, irrespectively to the marker class. Two linkage maps were produced, one for each parent. The female map had 203 loci, of which 160 (71 SSR and 89 AFLP) constituted the framework map at a LOD score ≥ 2.0. The map was 1,758.5 cM(K) long, covering 46% of the estimated genome length. The male map had only 143 loci, of which 116 (28 SSR, 87 AFLP and the sex determinant) constituted the framework map. The map length was only 1,104.1 cM(K), covering 34% of the estimate genome length. Only 35 SSR loci were mapped in the male parent because 18% of SSR loci that were characterised did not amplify in A. callosa, and 48% were homozygous. The choice of parents in the pseudo-testcross is critically discussed. The sex determinant was mapped in A. callosa.

We have isolated and sequenced microsatellites in several fruit crop species, such as grape (Vitis vinifera L.), kiwifruit (Actinidia, chinensis Planch), olive (Olea europea L.), and peach (Prunus persica (L.) Batsch), using microsatellite repeat-enriched genomic libraries. The efficiency of the enrichment procedure was variable (about 50 % of positive plaques in kiwifruit and peach, lower values in grape and olive). The re-amplification from the source genomic DNA was obtained in most cases. The polymorphism was high in all species assayed (5 to 15 alleles/locus in grape, 9 to 17 alleles/locus in kiwifruit, 2 to 8 alleles/locus in peach, data for olive are still being scrutinised). The Mendelian segregation was demonstrated for 8/10 SSR analysed in grape, 99/233 SSR analysed in kiwifruit, and 17/26 SSR analysed in peach. All microsatellites segregated as a single locus in peach, whereas in kiwifruit, grape, and olive several primer pairs amplified two loci each, in agreement with the suspected polyploid origin of the last species. The somatic stability was assayed in peach and olive, using three long-living accessions of cv. 'Redhaven' of different origin and 2-4 centuries-old plants of olive for each of the eight cultivars assayed. No pattern variation of the 26 microsatellites analysed was found in peach, whereas in olive rare intra-variety polymorphisms were observed in several of the 35 microsatellites analysed up to now. The assays on cross-species transportability were carried out in species representative of each genus (14 species of Vitis, 8 species of Actinidia, and 7 species of Prunus) and gave positive amplifications in high percentages (91 % of microsatellites amplified in all Vitis species, 52 % in all Prunus species, and 75 % in all Actinidia species). In some cases, successful amplifications were obtained also in related genera such as Parthenocissus, a genus close to Vitis, and Malus, which together with the genus Prunus belong to the Rosaceae family.

Eleven microsatellites isolated from grapevine (Vitis vinifera) were used to study the degree of conservation of these sequences across different Vitis species. Nine microsatellites were newly isolated, the remaining two (VVS2 and VVS5) came from the literature. A preliminary assay on the conservation of priming sites was carried out on 14 non-V. vinifera species, including relevant taxa for breeding. Parthenocissus quinquefolia was added as representative of a related genus. Cross-species amplification was obtained in 94% of the 176 genotype×locus tested combinations. Three microsatellite loci were then cloned and sequenced in ten species. The microsatellite repeat was found present in all cases. The repeat region was often longer in V. vinifera than in the other species. Furthermore the non-source species showed interruptions in the repeat. In spite of these constraints, which could reduce the polymorphism of microsatellites in non-source species, the results demonstrate the possibility of extending the use of microsatellite markers to wild germplasm and inter-specific hybrids. Point mutations have been found in microsatellite flanking regions and these variations have been used to investigate the genetic relationship among taxa. The Neighbor-joining tree that was obtained on the basis of ten nucleotide variations, showed that there is not a clear cut difference between American, Asian and European species and that the actual taxonomy which reflects the geographical distribution of species must most likely be revised. Moreover, in general, nucleotide variations which occur in microsatellite flanking regions provide new molecular tools for investigating the evolution of species.

We isolated and sequenced 26 microsatellites from two genomic libraries of peach cultivar 'Redhaven', enriched for AC/GT and AG/CT repeats, respectively. For 17 of these microsatellites, it was possible to demonstrate Mendelian inheritance. Microsatellite polymorphism was assayed in 50 peach and nectarine cultivars. Of the 1300 PCRs carried out, all but two produced amplified products of the expected size. All microsatellites were polymorphic, showing 2-8 alleles per locus. Heterozygosity ranged from 0.04-0.74 (mean 0.47); the discrimination power (PD) ranged from 0.04-0.84 (mean 0.60). Cultivar heterozygosity varied greatly, with one cultivar ('Independence') being homozygous at all loci. The set of microsatellites discriminated all cultivars investigated, except several sport mutations, i.e., 'Dixitime' vs. 'Springcrest', 'Compact Redhaven' vs. 'Redhaven', and two pairs of cultivars, 'Venus' vs. 'Orion' and 'Elegant Lady' vs. 'Rome Star', whose pedigrees are controversial. We were able to analyze the paternity of several cultivars. In most cases, the parenthood was confirmed. The comparison of three long-living 'Redhaven' accessions supplied by different repositories did not provide any evidence of somatic instability of microsatellites. Hence, microsatellites, ranked according to their information content, are recommended as markers of choice for peach fingerprinting and suggestions are provided for interpreting band profiles and the correct sizing of alleles.Key words: genetics, molecular markers, paternity analysis, pedigree analysis, simple sequence repeat.

An α-amylase gene product was isolated from apple fruit by reverse-transcriptase PCR using redundant primers, followed by 5′ and 3′ RACE. The gene is a member of a small gene family. It encodes a putative 46.9 kDa protein that is most similar to an α-amylase gene from potato (GenBank accession M79328). In apple fruit this new gene was expressed at low levels, as detected by reverse-transcriptase PCR, in a number of plant tissues and during fruit development. Highest levels of mRNA for this transcript were observed 3 to 9 days after placing apple fruit at 0.5 °C. Phylogenetic analysis of amino acid sequence places the potato and apple proteins as a distinct and separate new subgroup within the plant α-amylases, which appears to have diverged prior to the split between monocotyledonous and dicotyledonous plants. These two divergent α-amylases lack the standard signal peptide structures found in all other plant α-amylases, and have sequence differences within the B-domain and C-domain. However, comparisons with structures of known starch hydrolases suggest that these differences are unlikely to affect the enzymatic α-1,4-amylase function of the protein. This is the first report of upregulation of a dicotyledonous α-amylase in response to low temperature, and confirms the presence of a new family of α-amylases in plants.