Fruit Tree Breeding Research Articles

Biotech/GM crops in horticulture: plum cv. HoneySweet resistant to Plum pox virus

Commercialisation of Biotech/GM (Biotech) crops started in 1995. Not only field crops, but also horticultural transgenic crops are under development and are beginning to be commercialised. Genetic engineering has the potential to revolutionise fruit tree breeding. The development of transgenic fruit cultivars is in progress. Over the past 20 years an international public sector research team has collaborated in the development of HoneySweet plum which is highly resistant to Plum pox virus (PPV) the most devastating disease of plums and other stone fruits. HoneySweet was deregulated in the USA in 2010. HoneySweet (aka C5) has been evaluated for eleven years (2002–2012) in a regulated field trial in the CzechRepublic for the resistance to PPV, Prune dwarf virus (PDV), and Apple chlorotic leaf spot virus (ACLSV), all of them being serious diseases of plum. Even under the high and permanent infection pressure produced through grafting, PPV has only been detected in HoneySweet trees in several leaves and fruits situated close to the point of inoculum grafting. The lack of infection spread in HoneySweet demonstrates its high level of PPV resistance. Co-infections of PPV with PDV and/or ACLSV had practically no influence on the quantity and quality of HoneySweet fruit which are large, sweet, and of a high eating quality. In many respects, they are superior to the fruits of the well-known cultivar Stanley. Many fruit growers and fruit tree nurseries in the CzechRepublic are supportive of the deregulation of HoneySweet plum to help improve the plum production and control the spread of PPV.

Characterization of the S-RNase genomic DNA allele sequence in Prunus speciosa and P. pseudocerasus

In this study, eight S-RNase alleles were isolated from two Prunus pseudocerasus and two Prunus speciosa accessions by PCR amplification from genomic DNA. Analysis of the amino acid sequences revealed five novel and three published S-alleles. These S-RNases share typical structural features with S-RNases from other Prunus species exhibiting gametophytic self-incompatibility. The deduced amino acid identities ranged from 60.8 to 75.6% among four S-RNase alleles in Prunus speciosa and ranged from 73 to 81.4% among four S-RNase alleles in Prunus pseudocerasus. The size of the first introns ranged from 197 to 341bp, and the size of second introns ranged from 81 to 1182bp. Sequence analysis demonstrated that the deduced amino acid identities, by comparison with other Prunus species, were often higher than those of intraspecific identities. Moreover, exceptionally high identities were found between Pspe-S1 and Pd-S28; between Pspe-S31 and Pm-S6; among Pspe-S51, Pa-S29 and Pweb-S7; and among Pps-S13, Psim-S4 and Ps-Sb, indicating that the S-RNase alleles evolved before Prunus species divergence. Interestingly, the similarities of the first and second introns were also high between the two S-RNase alleles, which range from 83.63 to 100% among the first introns and from 46.13 to 100% among the second introns. These information could not increase our knowledge on the S-alleles of Prunus species, but is available for molecular breeding of fruit trees, to avoid cross-incompatibility.

REVIEW OF FRUIT GENETICS AND BREEDING PROGRAMMES AND A NEW EUROPEAN INITIATIVE TO INCREASE FRUIT BREEDING EFFICIENCY

The European fruit industry is facing economic challenges imposed by increasingly fierce international competition and decreasing fruit consumption, societal demand for a more sustainable production, and biological problems caused by climate changes. Releasing new cultivars that meet these challenges is a major goal of all European breeding programmes. However, addressing them has been slow due to the nature of fruit tree breeding: long term, low efficiency and high cost. For the past 15 years, efficient networks of fruit geneticists and genomicists have progressively been built thanks in particular to EU-funded projects. Europe has thus become a leader in research on fruit genetics aimed at enhancing fruit quality traits as well as resistance to biotic stresses. European teams working on fruit genetics have developed up-to-date tools and skills covering most of the omics fields as well as statistics and software development. Although fruit breeding is very active in Europe, very few breeding programmes are really using the output of the fruit genetics/genomics research. A few bottlenecks can explain this situation. To solve that, a new European initiative has been set up by the FruitBreedomic. This large collaborative project, has the strategic goal of improving the efficiency of current fruit breeding programmes by bridging the existing gap between molecular genetics research and application in breeding.

Environmental Variance Components of Fruit Ripening Date as Used in Both Phenotypic and Marker-assisted Selection in Japanese Pear Breeding

The effectiveness of detected quantitative trait loci (QTLs) and molecular markers associated with them in tree fruit breeding is measured by the percentages of the variance associated with detected QTL effects accounting for not phenotypic variance, but genetic variance of the trait. The genetic variance can be obtained by subtracting environmental variance from the phenotypic variance. Once accurate environmental variance components are obtained for a given selection field, environmental variances under any number of replications and measurement repetitions can be estimated. We estimated environmental variance components of fruit ripening date measured by days in a Japanese pear (Pyrus pyrifolia Nakai) breeding field in the National Institute of Fruit Tree Science, Tsukuba, Ibaraki, Japan. We estimated variance among fruits within a tree (σf2) as 25.6, among trees within a genotype (σt2) as 0.2, among years (σy2) as 9.4, associated with genotype × year interaction (σgy2) as 7.9, and associated with tree × year interaction (σty2) as 1.2. Because σf2 was the largest environmental variance component, increasing the number of fruit evaluated would most effectively reduce the environmental variance, and tree replication would not because of very small σt2 and σty2. The 95% confidence limit of a genotypic value was ± 10 days in the evaluation of five fruits on a single tree in a year and ± 7 days over 2 years. Broad-sense heritability in a family, each offspring in which was evaluated using five fruits on a single tree in a single year, was estimated at 0.83 for three full-sib families analyzed.

Intellectual Property and Horticultural Science: The Path from Idea, Technology Development, Commercialization, and Impact

Intellectual Property and Horticultural Science: The Path from Idea, Technology Development, Commercialization, and Impact

Population structure and genetic bottleneck in sweet cherry estimated with SSRs and the gametophytic self-incompatibility locus

BackgroundDomestication and breeding involve the selection of particular phenotypes, limiting the genomic diversity of the population and creating a bottleneck. These effects can be precisely estimated when the location of domestication is established. Few analyses have focused on understanding the genetic consequences of domestication and breeding in fruit trees. In this study, we aimed to analyse genetic structure and changes in the diversity in sweet cherry Prunus avium L.ResultsThree subgroups were detected in sweet cherry, with one group of landraces genetically very close to the analysed wild cherry population. A limited number of SSR markers displayed deviations from the frequencies expected under neutrality. After the removal of these markers from the analysis, a very limited bottleneck was detected between wild cherries and sweet cherry landraces, with a much more pronounced bottleneck between sweet cherry landraces and modern sweet cherry varieties. The loss of diversity between wild cherries and sweet cherry landraces at the S-locus was more significant than that for microsatellites. Particularly high levels of differentiation were observed for some S-alleles.ConclusionsSeveral domestication events may have happened in sweet cherry or/and intense gene flow from local wild cherry was probably maintained along the evolutionary history of the species. A marked bottleneck due to breeding was detected, with all markers, in the modern sweet cherry gene pool. The microsatellites did not detect the bottleneck due to domestication in the analysed sample. The vegetative propagation specific to some fruit trees may account for the differences in diversity observed at the S-locus. Our study provides insights into domestication events of cherry, however, requires confirmation on a larger sampling scheme for both sweet cherry landraces and wild cherry.

ENABLING MARKER-ASSISTED SEEDLING SELECTION IN THE WASHINGTON APPLE BREEDING PROGRAM

Breeding new cultivars is a slow and expensive process, but genetic screening tools exist to improve efficiency. We employed the marker-assisted breeding (MAB) Pipeline approach to facilitate routine use of MAB in the Washington apple breeding program. This study traced the journey of one marker-locus-trait (M-L-T) association from publication through high-throughput PCR-based genetic testing to application. The final stages of the Pipeline involve determining cost-efficient highthroughput marker-assisted seedling selection (MASS) schemes with available genetic tests, followed by a trial run on several thousand seedlings. For maximum cost-efficiency in the Washington apple breeding program, genetic screening should be implemented prior to expensive grafting and field planting operations but usually after inexpensive visual culling for traits such as disease susceptibility. Routine MAB is now enabled for this tree fruit breeding program and we estimate that at least 60% of conventional operating costs for first-stage seedling selection could be saved using the available genetic test.

Principles and practices of plant genomics. Volume 2: Molecular breeding

Principles and practices of plant genomics. Volume 2: Molecular breeding

Resistance Gene Analogs (RGA) as a tool in fruit tree's breeding

Breeding for pest and disease resistance comes as a major objective behind the fruit traits. To increase the effectiveness of fruit resistance breeding application of the Marker Assisted Selection ( MAS) is advantageous. For generating molecular markers which enable the following of interesting traits basically two methods are available: targeted marker design based on conservative region of already known Resistance ( R) gene sequences or randomly generated markers. The creation and the application of these homology based markers are the object of this review in the main temperate zone fruit species.

Induction of Seedlessness in Citrus: From Classical Techniques to Emerging Biotechnological Approaches

Seedlessness can be obtained through parthenocarpy (i.e., fruit formation without fertilization or embryo abortion). In practice, the actual reduction in seed number in parthenocarpic plants is often exaggerated by coupling parthenocarpy with self-incompatibility or male sterility. Traits related to seedlessness, such as parthenocarpy, can be introduced into genetic accessions through conventional cross-breeding. However, conventional breeding in Citrus L. species faces several limitations. First, these species have long juvenile (nonflowering) periods during which a relatively thick canopy develops, which limits the size of seedling populations that can be maintained for further evaluation. Second, they have a narrow genetic base, which limits the availability of alternative alleles that could be introgressed into other lines for the formation of a particular phenotype such as parthenocarpy. Third, breeding efforts are limited by the lack of knowledge of the mode of inheritance of specific characteristics. Fourth, and last, breeding efforts are limited by the polygenic nature of many important traits. Despite these limitations, conventional breeding in fruit trees has yielded improved cultivars and will most likely continue to be a very important strategy. However, emerging biotechnological approaches should be continuously evaluated for their potential for expediting such breeding efforts. The objective of this review is to present, evaluate, and discuss conventional and emerging biotechnological approaches for the induction and maintenance of seedlessness in a variety of crops. Particular attention will be paid to citrus crops, including the presentation and discussion of some preliminary data on the genetic inheritance of parthenocarpy.

Constitutive expression of two apple (Malus × domestica Borkh.) homolog genes of LIKE HETEROCHROMATIN PROTEIN1 affects flowering time and whole-plant growth in transgenic Arabidopsis

Fruit trees, such as apple (Malus x domestica Borkh.), are woody perennial plants with a long juvenile phase. The biological analysis for the regulation of flowering time provides insights into the reduction of juvenile phase and the acceleration of breeding in fruit trees. In Arabidopsis, LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is involved in epigenetic silencing of the target genes such as flowering genes. We isolated and characterized twin apple LHP1 homolog genes, MdLHP1a and MdLHP1b. These genes may have been generated as a result of ancient genome duplication. Although the putative MdLHP1 proteins showed lower similarity to any other known plant LHP1 homologs, a chromo domain, a chromo shadow domain, and the nuclear localization signal motifs were highly conserved among them. RT-PCR analysis showed that MdLHP1a and MdLHP1b were expressed constantly in developing shoot apices of apple trees throughout the growing season. Constitutive expression of MdLHP1a or MdLHP1b could compensate for the pleiotropic phenotype of lhp1/tfl2 mutant, suggesting that apple LHP1 homolog genes are involved in the regulation of flowering time and whole-plant growth. Based on these results, LHP1 homolog genes might have rapidly evolved among plant species, but the protein functions were conserved, at least between Arabidopsis and apple.

Negative inbreeding effects in tree fruit breeding: self-compatibility transmission in almond

Inbreeding depression has been observed in most fruit trees, negatively affecting the offspring of related parents. This problem is steadily increasing due to the repeated utilization of parents in breeding programmes. In almond, self-compatibility transmission from 'Tuono' to its offspring remains partially unexplained due to deviations from the expected genotype ratios. In order to test if these deviations could be due to inbreeding, the S-genotypes of the seedlings of four almond families, 'Tuono' (S(1)S(f )) x 'Ferragnès' (S(1)S(3)), 'Tuono' (S(1)S(f)) x 'Ferralise' (S(1)S(3)) and reciprocal crosses were studied. The S-genotype determination of each seedling by separation of stylar S-RNases and by S-allele-specific PCR amplification gave identical results. The ratio of S-genotypes of the family 'Tuono' x 'Ferralise' was the one least adjusted to the expected 1:1 ratio, because the number of self-compatible seedlings (S(f)S(3)) was less than a half the number of self-incompatible ones (S(1)S(3)). A mechanism acting against inbreeding would favour cross-breeding in the following generation to increase heterozygosity. This fact stresses the need to avoid crosses between related parents in fruit breeding programmes.

BPMADS4 - A MADS BOX GENE OF BIRCH INDUCES FLOWERS ON TRANSGENIC APPLE PLANTS IN VITRO

In comparison to herbaceous plants, the breeding of fruit trees, such as apple (Malus x domestica Borkh.), is more time-consuming due to its long generation time. Shortened juvenility and precocious flowering are, therefore, important breeding goals. Flower initiation has been intensively studied in Arabidopsis and orthologues/ homologues of LEAFY (LFY), APETELA1 (AP1), and TERMINAL FLOWER (TFL1) have been cloned from apple. One approach to shortening the juvenile phase of perennial crops is to reduce juvenility/vegetative maintenance factors, such as TFL1, by gene silencing. An alternative approach is to induce flowering by the over-expression of genes involved in the formation of floral meristems. In this context, the bpMADS4 gene of silver birch, which is similar to FRUITFULL of Arabidopsis, was overexpressed in apple. This gene is normally expressed in the apical meristem before the appearance of meristems (Elo et al., 2001). In transgenic apple, the expression of bpMADS4 was controlled by the constitutive CAMV35S promoter. Twenty transgenic lines were obtained after Agrobacterium-mediated gene transfer into apple. The integration and expression of the nptll and bpMADS4 genes were characterized by PCR analysis and RT-PCR. To quantify the level of transgene transcription a Real Time PCR procedure was carried out. Solitary flowers were detected on in vitro shoots of line T1165 thirteen weeks after transformation. Four other lines also produced flowers during in vitro propagation. The initial flowers were solitary, but shoots later developed clusters with about five flowers. Most of these flowers appeared morphologically normal but some were abnormal. In spring 2005, shoots of these lines were rooted and transferred to the greenhouse for further evaluation. To our knowledge, this is the first report of in vitro flower induction in transgenic apple.

DEREGULATION OF PLUM POX RESISTANT TRANSGENIC PLUM 'HONEYSWEET'

Genetic engineering (GE) has the potential to revolutionize fruit tree breeding. It is an approach that can specifically target genetic improvements and allow for the development of novel, useful traits. While GE does not provide a panacea for all of the difficulties associated with fruit tree breeding, it can be a useful approach to increase the efficiency and effectiveness of breeding programs. In spite of the potential utility of GE for fruit tree improvement, the technology has not, to date, been widely exploited in these species. Of over 11,000 field tests of transgenic plants in the United States between 1987 and 2004, less than 1% have involved fruit tree species. Transgenic plum trees that are highly resistant to Plum pox virus (PPV) are one example of GE that can be of significant benefit to growers and consumers while providing unique genetic material for use in conventional breeding programs. The development and testing of this plum has spanned 15 years and included researchers in five countries. Currently this plum, 'HoneySweet', is being evaluated for deregulation by U.S. regulatory agencies so that it may possibly be released in the future to breeders and growers who are concerned about the threat of PPV to U.S. stone fruit production.

INTEGRATING GENOMICS INTO ROSACEAE FRUIT BREEDING

The amount of information on genomic resources for Rosaceae species has rapidly grown since the beginning of this century. A genome database for Rosaceae (GDR) is now available, with information from EST sequences, markers, linkage maps and a peach physical map. One of the key resources for this family is a high-density reference map for stone fruit (Prunus) constructed entirely from transferable markers (mainly RFLPs and SSRs). Markers from this map have been used to construct maps in seven Prunus species, and comparisons of these maps have established that they all share the same genome. Apple and pear also have highly syntenic genomes, and the data available on the comparison between peach and apple show an incomplete but high level of synteny. The comparisons of Prunus with strawberry and rose are now the targets to improve our understanding of the Rosaceae genome. Maps have been used to determine the genome positions of major genes and QTLs for important agronomic characters, and have provided markers for selection of these characters. Because of the syntenic genome of Prunus, 28 major genes and 28 QTLs mapped in different species have been placed on a single consensus map. Co-locations of genes and QTLs among Prunus species have been found, suggesting that the information of one species could be useful for others. Some of this information is already used for rootstock and scion breeding programs. Whole-genome searches of candidate genes, based on the growing transcriptome information is already producing results, improving our knowledge of disease resistance and fruit quality characters, which is likely to be useful for fruit tree breeding.

Antisense Expression of MdTFL1, a TFL1-like Gene, Reduces the Juvenile Phase in Apple

Because fruit trees such as apple (Malus ×domestica Borkh.) flower and set fruit only after an extended juvenile phase lasting several years, efficient breeding of fruit trees is limited. We previously suggested that MdTFL1 (Malus ×domestica TFL1) functions analogously to TERMINAL FLOWER 1 (TFL1) and that MdTFL1 is involved in the maintenance of the juvenile/vegetative phase in apple. To clarify the function of MdTFL1 in apple, we produced transgenic `Orin' apple trees expressing MdTFL1 antisense RNA. One of them flowered only 8 months after the transfer to the greenhouse, whereas the nontransformed control plants have not flowered in nearly 6 years. As expected, the expression of endogenous MdTFL1 was suppressed in the transgenic lines that showed precocious flowering. In addition, the expression level of the transgene was correlated with the reduction of the juvenile phase. These findings confirm that MdTFL1 functions like TFL1 and that MdTFL1 maintains the juvenile and vegetative phase in apple. Flower organs of the transgenic apple trees were normal in appearance, and a precocious flowering transgenic line set fruit and seeds. Interestingly, some flowers of the transgenic apple trees developed without undergoing dormancy. The expression of MdTFL1 in apple may affect flower development as well as flower induction.

Transformation of fruit trees. Useful breeding tool or continued future prospect?

Regeneration and transformation systems using mature plant material of woody fruit species have to be achieved as a necessary requirement for the introduction of useful genes into specific cultivars and the rapid evaluation of resulting horticultural traits. Although the commercial production of transgenic annual crops is a reality, commercial genetically-engineered fruit trees are still far from common. In most woody fruit species, transformation and regeneration of commercial cultivars are not routine, generally being limited to a few genotypes or to seedlings. The future of genetic transformation as a tool for the breeding of fruit trees requires the development of genotype-independent procedures, based on the transformation of meristematic cells with high regeneration potential and/or the use of regeneration-promoting genes. The public concern with the introduction of antibiotic resistance into food and the restrictions due to new European laws that do not allow deliberate release of plants transformed with antibiotic-resistance genes highlight the development of methods that avoid the use of antibiotic-dependent selection or allow elimination of marker genesfrom the transformed plant as a research priority in coming years.

Relocating Large Trees for Germplasm Conservation in Tree Fruit Breeding Programs

Mature seedling trees of pear (Pyrus communis and interspecific hybrids), and fruiting trees of peach and nectarine (Prunus persica), apricot (Prunus armeniaca), and pear were relocated during the dormant season using tree spades. During the growing season immediately following, some signs of drought stress were noticed but all trees grew well enough that they could be used as a source of budwood for limited propagation purposes. When drip irrigation was supplied, supplemented by overhead irrigation as required, normal growth and production resumed within two growing seasons of the move. Some tree losses (less than 10% of trees moved) were reported from one site where the soil type was Fox sand with very poor water holding capacity. These tree losses were attributed to an inadequate water supply to the root ball, even though the site was irrigated. Our experience has demonstrated the feasibility of relocating relatively large trees, which can be beneficial for germplasm conservation in a tree fruit breeding program. However, it is probably not economically viable to relocate such trees for commercial production.

MANGO BREEDING AND THE POTENTIAL OF MODERN BIOLOGY

Classical breeding of fruit trees is a rather inefficient and slow procedure. However, all current mango cultivars result from either random selection or classical breeding. The current interesting mango selections originating from the Israeli breeding project include: 'Shelly', a round, colored, good tasting fruit with a long shelf-life and 'Naomi' an attractive red-orange colored fruit with a delicate taste. One of the modern breeding methodologies is based on the application of DNA markers, construction of genetic maps and identification of linkage between DNA markers and the genes of interest. Currently, we are trying to discover SNPs both in-silico and by direct sequencing and to genotype them using the MassArray method. We are applying SNPs in tomato for the assessment of biodiversity and for gene hunting. We have applied AFLP to study the genetic relationship among 16 mango cultivars and seven mango rootstocks and for the construction of a genetic map. The average Band-Sharing for mango is 81%. The preliminary map which was constructed using the progeny of a cross between 'Keitt' and 'Tommy-Atkins' consists of 13 linkage groups, defined by 34 AFLP polymorphic bands and covers about 160 centi-Morgans. Future developments of horticultural crops are expected to be based on molecular biology and genomics. Examples of modern technologies as well as examples of biotechnological applications in plants will be discussed.

SUCCESSFUL ESTABLISHMENT OF IN VITRO CULTURES OF PRUNUS CERASIFERA HYBRIDS BY EMBRYO CULTURE OF IMMATURE FRUITS

Rapid obtaining of clonal plants is desirable to shorten crossing programs in fruit tree breeding. In this work, seedling germination and multiplication in vitro allowed us to establish successful in vitro cultures of hybrid seedlings. As a part of a breeding program in apricot rootstocks, we have obtained interespecific hybrid seeds after cross-pollination: myrobalan x apricot (Prunus cerasifera x armeniaca). Immature fruits were harvested and embryos, isolated in aseptic conditions, were grown in culture medium. From a total of 419 seeds, we obtained a germination rate of 81.4%, succeeding in the rescue of 73% of aborting seeds. The successful micropropagation and acclimatization methods applied, yielded a high number of cloned plants from every cultured embryo, allowing us to obtain soil-established plants in a very short time. Advantages of in vitro germination and micropropagation versus conventional methods have been assessed from the point of view of the length of the process and the number of clonal plants obtained.