Apples, originating from Central Asia, are widely cultivated worldwide, with China producing 47.6 million tonnes annually. Despite their global importance, breeding programs often focus on a limited number of high-quality cultivars, potentially threatening genetic diversity. To mitigate this risk, advanced statistical methods could be employed to improve selection strategies. For example, the multi-trait BLUP method, which accounts for genetic correlations among traits, can reduce selection bias but increases computational complexity due to the model’s intricacy and slow convergence of the REML process. Bayesian methods, like Markov Chain Monte Carlo (MCMC), offer a solution to these challenges. This study compares single-trait and multi-trait approaches using REML/BLUP and MCMC/BLUP to estimate variance components and predict genetic values. Over two seasons, phenotypic data from 304 seedlings and 16 parents were analyzed using an animal model to predict their genetic values related to fruit quality traits. Significant progress was made in estimating genetic parameters and selecting parents for traits such as fruit weight, flesh firmness, and soluble solids content in the University of Udine’s apple breeding program. The multi-trait BLUP method improved the accuracy of predicted breeding values, particularly for fruit weight, polar diameter, flesh firmness, and soluble solids content. The multivariate repeated measures model, despite low correlations between some trait pairs, was recommended for integrating multi-season results and accounting for trait correlations. The Bayesian MCMC approach proved superior in genetic evaluations, offering higher heritabilities and genetic gains compared to Fisherian methods (REML). It effectively handles small sample sizes, captures more genetic variance, and enhances breeding value predictions, recommending traits like fruit size, firmness, and sweetness for selecting superior parents.

The DNA fingerprinting of fruit crops, based on DNA microsatellite markers that are considered to be the key markers for the molecular analysis of germplasm collections, is reviewed. Simple sequence repeats (SSRs) remain the markers of choice for fingerprinting in humans, animals, plants, and other living organisms. This review, that considers 44 fruit species, provides a set of markers that are suitable for profiling accessions and that should make the databases produced in different laboratories more comparable. Every effort has been made to select SSR markers that are robust and easily scorable considering that such analyses are sometimes used in legal cases. The review first describes the basic protocols, procedures, and methods of data analyses; it then describes the fingerprinting of individual species or groups of species, providing a set of SSR markers and appropriate guidance based on the revised literature and on the authors' experience.

CREA Research Centre for Viticulture and Enology, viale XXVIII Aprile 26, 31015, Conegliano (TV), Italy INRAE, Université de Strasbourg, UMR 1131, 28 rue de Herrlisheim, 68000, Colmar, France Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, via delle Scienze 206, 33100, Udine (UD), Italy ______________________________________________________________________________

This contribution considers the kiwifruit industry in Italy, taken as a "case study" and the main aspects that are characterizing the crop second only to China in world importance. The main points addressed are: planting areas and production trends; changes in the cultivars grown; emerging diseases and their consequences in the pre- and postharvest crop management; finally, the Club and Consortia that are influencing the national industry. Considering what we should expect in the future and what action should be taken to maintain the leading position that Italy has reached we need to consider two main issues: a political and a technical one. As regards the political issue: the most important countries have now consolidated their choices and strategies. We need to make competitive the national industry by taking a further step towards common goals. The second issue concerns technical aspects: is the kiwifruit industry ready for a completely different and innovative agricultural approach, new training systems, control of all the production factors that could allow a production significantly higher than that we have achieved so far?

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.

Kiwifruit belong to the genus Actinidia with 54 species apparently all functionally dioecious. The sex-determinants of the type XX/XY, with male heterogametic, operate independently of the ploidy level. Recently, the SyGI protein has been described as the suppressor of female development. In the present study, we exploited the CRISPR/Cas9 technology by targeting two different sites in the SyGI gene in order to induce a stable gene knock-out in two tetraploid male accessions of Actinidia chinensis var. chinensis. The two genotypes showed a regenerative efficiency of 58% and 73%, respectively. Despite not yet being able to verify the phenotypic effects on the flower structure, due to the long time required by tissue-cultured kiwifruit plants to flower, we obtained two regenerated lines showing near fixation of a unique modification in their genome, resulting in both cases in the onset of a premature stop codon, which induces the putative gene knock-out. Evaluation of gRNA1 locus for both regenerated plantlets resulted in co-amplification of a minor variant differing from the target region for a single nucleotide. A genomic duplication of the region in proximity of the Y genomic region could be postulated.

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

EDITORIAL article Front. Plant Sci., 15 July 2021Sec. Plant Development and EvoDevo https://doi.org/10.3389/fpls.2021.719588

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.

Nutritional unbalances, such as calcium deficiency at the fruit level, are generally the causative agent of post-harvest disorders in apples. Foliar application of Ca as calcium chloride is the current solution to increase Ca concentration in apples, even though the effectiveness of this approach is often not satisfactory. In this research, we tested the efficacy of a combined application of Ca with selected biostimulants to improve apple quality and to reduce the incidence of storage disorders. The experiment was conducted in two “Jonathan” apple orchards that differed in management systems and characteristics. Tree canopies were sprayed with calcium chloride alone and in combination with a commercial product containing zinc and silicon or a seaweed extract. The seaweed extract increased apple quality by boosting the reddish coloration (+32% of color index) and by enhancing final anthocyanin concentration of fruit skin. Both biostimulants significantly reduced (by 20%) the incidence of the physiological disorder, known as “Jonathan spot”, after 160 days of storage. Increased concentration of nutrients (Ca, Zn, and Mn) in the skin of apples after biostimulant applications, together with changes of the phenolic profile during the storage, are discussed as the possible causes of the reduced fruit susceptibility to post-harvest disorders.

Ancient germplasm represents a reservoir of traits and genes that might maintain large the genetic diversity of a species, like pear, and help breeders to cope with climatic changes and the evolving demand of the market. This paper reports the analysis of 170 pear accessions profiled at 12 simple sequence repeat (SSR) markers. The collection was replicated in three different locations and the identification of matching profiles of duplicated genotypes reduced the dataset to 118 genotypes. Of these, 32 (27.1%) were shown to be triploid by flow cytometry, a result that was confirmed by the occurrence of triallelic profiles at molecular markers and the large leaf size. The geographic location of original plants explained very little molecular variance (2%), that was instead very high among genotypes (98%). This evidence confirms the extensive exchange of plant material among the different geographic areas of the region as well as with neighbouring countries as reported in historical documents. The analysis of synonymy revealed 38 duplicated genotypes and reduced the dataset to 80 unique genotypes, of which 12 were triploid (15.0%). The molecular metrics analysed on 70 unique diploid genotypes, including two commercial cultivars, ‘Abate Fétél’ and ‘Conference’ added as controls, revealed that the observed heterozygosity over all loci (Ho) was 0.742 (range 0.471–0.937 for individual loci), while the expected heterozygosity or gene diversity was 0.808 (range 0.534–0.912). The polymorphic information content was 0.784 on average (range 0.501–0.906 for individual loci). The probability of identity (PID) calculated for individual loci and unrelated genotypes ranged from 0.014 for the most discriminating locus, Ch01d09, to 0.250 for the least discriminating one, Ch04e03. The PID product over all loci was 2.50e−16 for unrelated genotypes and 4.86e−06 for full sibs. The analysis of data is integrated with a discussion on the use of SSR markers in pear genotyping and the origin and the frequency of triploids compared with data from the literature. Finally, a discussion on the resemblance of several groups of cultivars to those of the pomological treatises is included.

This paper reports the genetic diversity of apple germplasm collected in the Friuli Venezia Giulia region (northeastern 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.

Sharka, a common disease among most stone fruit crops, is caused by the Plum Pox Virus (PPV). Resistant genotypes have been found in apricot (Prunus armeniaca L.), one of which—the cultivar ‘Lito’ heterozygous for the resistance—has been used to map a major quantitative trait locus (QTL) on linkage group 1, following a pseudo-test-cross mating design with 231 individuals. In addition, 19 SNP markers were selected from among the hundreds previously developed, which allowed the region to be limited to 236 kb on chromosome 1. A ‘Lito’ bacterial artificial chromosome (BAC) library was produced, screened with markers of the region, and positive BAC clones were sequenced. Resistant (R) and susceptible (S) haplotypes were assembled independently. To refine the assembly, the whole genome of ‘Lito’ was sequenced to high coverage (98×) using PacBio technology, enabling the development of a detailed assembly of the region that was able to predict and annotate the genes in the QTL region. The selected cultivar ‘Lito’ allowed not only to discriminate structural variants between the two haplotypic regions but also to distinguish specific allele expression, contributing towards mining the PPVres locus. In light of these findings, genes previously indicated (i.e., MATHd genes) to have a possible role in PPV resistance were further analyzed, and new candidates were discussed. Although the results are not conclusive, the accurate and independent assembly of R and S haplotypes of ‘Lito’ is a valuable resource to predict and test alternative transcription and regulation mechanisms underpinning PPV resistance.

Grape is one of the most important fruit crops cultivated worldwide both for fresh consumption and wine production. All cultivars of Vitis vinifera are susceptible to several pathogens. Downy mildew (Plasmopara viticola) is one of the most dangerous diseases, causing heavy defoliation and crop losses. In the southern Mediterranean countries, powdery mildew (Uncinula necator) is also of major concern. Grape cultivation accounts for a large part of the fungicides sprayed in Europe. An alternative would be to use grape cultivars resistant to pathogens. Here, we present a breeding program, developed at the University of Udine, that aims to pyramidize genes involved in the resistance to powdery and downy mildew into a high-quality genetic "vinifera background" for the selection of new wine and table grape cultivars. Almost 6000 seedlings resulting from 71 different crosses done during 2010-2013 were grown. The parents were selected from a panel of elite wine or table cultivars and resistant genotypes. Offspring with pyramidized genes were obtained from 64 of the initial crosses. Natural infections were recorded during the first year of field growth. Seedlings (820) were also genotyped using molecular markers linked to four recognized resistance genes to downy mildew (Rpv1, Rpv3, Rpv10, Rpv12) and four resistance genes to powdery mildew (Run1, Ren1, Ren3, Ren4). After phenotypic and genotypic selection, 1128 progenies were grafted onto 'SO4' rootstock and grown in the open field. The following traits were observed for at least 2 years of production: berry color, type of cluster (loose vs. compact), production (high, medium, low), vegetative habit (weak vs. vigorous), and susceptibility to diseases other than powdery and downy mildews (mainly Botrytis cinerea and black rot). A general overview of genotypes with pyramidized resistance is presented.

Key messageThe developmental morphology of male and female kiwifruit flowers is tracked to delimit a framework of events to aid the study of divergence in floral gene expression.AbstractThe transition from hermaphrodite to unisexual development of kiwifruit (Actinidia chinensis Planch) flowers has been reported previously, but differences in gene expression controlling sexual development for this species have not been associated with the major developmental changes occurring within pistils. We investigated the key stages in male and female flower development to define the point at which meristematic activities diverge in the two sexes. A combination of scanning electron microscopy and light microscopy was used to investigate pistil development from the earliest stages. We identified seven distinct stages characterized by differences in ovary size and shape, macrosporogenesis, ovule primordium development, anther locule lengthening, microspore wall thickening, and pollen degeneration. Sex differences were evident from the initial stage of development, with a laterally compacted gynoecium in male flowers. However, the key developmental stage, at which tissue differentiation clearly deviated between the two sexes, was stage 3, when flowers were 3.5 to 4.5mm in length at approximately 10 d from initiation of stamen development. At this stage, male flowers lacked evident carpel meristem development as denoted by a lack of ovule primordium formation. Pollen degeneration in female flowers, probably driven by programmed cell death, occurred at the late stage 6, while the final stage 7 was represented by pollen release. As the seven developmental stages are associated with specific morphological differences, including flower size, the scheme suggested here can provide the required framework for the future study of gene expression during the regulation of flower development in this crop species.

A study was conducted on 14 accessions of Actinidia spp. to evaluate their susceptibility to the bacterial canker. Experimental inoculations were carried out, under controlled conditions, on young leaves of kiwifruit plants grown in pots by spraying a calibrated water suspension of an Italian, virulent strain (isolated in 2013) of Pseudomonas syringae pv. actinidiae. Actinidia chinensis 'Belen' was used as positive control, being a known susceptible genotype. Plants inoculated with sterile distilled water and non-inoculated ones were used as negative control. The disease assessments were performed 21 days after inoculation with the pathogen. The disease severity (mean of leaf spots per treatment) allowed to detect one A. chinensis selection showing low susceptibility, comparable to that of selections belonging to A. arguta, A. hemsleyana and A. eriantha species, that showed a very low susceptibility. No selection was found more susceptible than 'Belen' but among all selections, one resulted comparable with it. Eight selections were found to be moderately susceptible, and three of them (A. chinensis selections) were less susceptible than A. deliciosa 'Hayward', that is known having an intermediate susceptibility.

Fruit quality is a key criterion used to select new cultivars in kiwifruit breeding programs. The aim of this research was to identify quantitative trait loci (QTLs) that control traits related to fruit quality in an F1 population derived from the cross of 'C8' (female) and 'A54.19' (male) diploid kiwifruit. A small female progeny was evaluated over 3 years for several traits: fruit weight, absorbance of chlorophyll (IAD) values of the skin and flesh, flesh firmness, soluble solids content (SSC), titratable acidity (TA), pH, dry matter (DM) content and ripening time (RT). Single or multiple QTL mapping models were applied separately for each year and all years combined. As a result, a total of 24 QTLs were detected on 12 different linkage groups (LGs). Some QTLs, such as flesh firmness, skin and flesh chlorophyll absorbance, are reported for the first time in kiwifruit. However, QTLs were not consistent among years, and were rarely identified in both parents in corresponding LGs. These results were expected, because of the limited size of the progeny. This work was the first attempt to identify genomic regions that control fruit-quality traits in the mapping cross population. Extension of the cross population to about 400 female individuals has been undertaken, and this will guarantee a more reliable and robust identification of QTLs related to fruit quality in future analyses.

Italian university farms provide a crucial support for teaching, research and innovation transfer in the agro-food, forestry, environmental and even social sectors. In order to point out their current values and weaknesses, and to start reflecting on possible development strategies, a survey was realised. A questionnaire was sent to the 25 Italian universities providing courses in the agro-food and forestry sectors. The main weaknesses emerged concern the availability of the required data, in particular economic data, and the economic sustainability of these farms, especially with regard to staff costs. Moreover, the survey made it possible to outline for the first time a cognitive framework of the Italian university farms, thus creating a useful database for future updates and insights.

Analysis of genetic components of variance in controlled mating designs allows estimation of the heritability of traits, the combining ability of cross parents and, in turn, their breeding value. The present paper report the analysis of a North Carolina model 2 mating design, where three female parents and 12 male parents were intercrossed, producing 35 cross families (one cross combination failed to produce seeds). The cross families, consisting of 40 to 142 offspring each, were observed in two subsequent years for time of bud break, time of flowering, flowers per cyme, flowering intensity, productivity, fruit weight, soluble solids content (SSC), flesh firmness, and flesh color. The genetic components of variance were computed using restricted maximum likelihood (REML) and best linear unbiased prediction (BLUP) on the R environment with the ASReml-R package. The narrow-sense heritability (h(2)) was low (< 0.30) for flowering time, flowers per cyme, flowering intensity, and productivity; intermediate (0.30-0.60) for time of bud break, fruit weight, SSC, and flesh color; and high (>0.60) for flesh firmness as an index of ripening time. Parents and secondary parents were ranked according to their breeding value for the main traits and the best parents as well the best parental combinations were also ranked for both productivity and fruit weight. The paper concludes by commenting on the importance of the evaluation of breeding value carried out on male genotypes, in which fruit-related traits cannot be phenotypically evaluated.

Background: Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models. Results: A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within 'Hongyang' The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned 'Hort16A' cDNAs and comparing with the predicted protein models for Red5 and both the original 'Hongyang' assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised 'Hongyang' annotation, respectively, compared with 90.9% to the Red5 models. Conclusions: Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.