McIntosh Wijcik Research Articles

Molecular Mechanisms Regulating the Columnar Tree Architecture in Apple

The columnar apple cultivar ‘McIntosh Wijcik’ was discovered as a spontaneous mutant from the top of a ‘McIntosh’ tree in the early 1960s. ‘McIntosh Wijcik’ exhibits the columnar growth phenotype: compact and sturdy growth, short internodes, and very few lateral shoots. Classical genetic analysis revealed that the columnar growth phenotype of ‘McIntosh Wijcik’ is controlled by a single dominant gene, Co. This review focuses on the advances made toward understanding the molecular mechanisms of columnar growth in the last decade. Molecular studies have shown that an 8.2 kb insertion in the intergenic region of the Co locus is responsible for the columnar growth phenotype of ‘McIntosh Wijcik’, implying that the insertion affects the expression patterns of adjacent genes. Among the candidate genes in the Co region, the expression pattern of MdDOX-Co, putatively encoding 2-oxoglutarate-dependent dioxygenase (DOX), was found to vary between columnar and non-columnar apples. Recent studies have found three functions of MdDOX-Co: facilitating bioactive gibberellin deficiency, increasing strigolactone levels, and positively regulating abscisic acid levels. Consequently, changes in these plant hormone levels caused by the ectopic expression of MdDOX-Co in the aerial organs of ‘McIntosh Wijcik’ can lead to dwarf trees with fewer lateral branches. These findings will contribute to the breeding and cultivation of new columnar apple cultivars with improved fruit quality.

A rapid real‐time PCR assay for detecting columnar growth in apples (Malus × domestica)

AbstractColumnar growth in apples can have considerable positive consequences for orchard maintenance, but the lack of columnar cultivars producing fruits with desired market quality prevents their broader utilization in orchards. New crossings focused on their breeding are therefore conducted throughout the world every year. Unfortunately, the reliable discrimination between columnar growth and normal phenotype is difficult until seedlings are 2–3 years of age. However, molecular marker–assisted selection (MAS) can be used in seedlings several weeks old providing immense help in breeding programmes focused on columnar apple production. Here, we describe a rapid and reliable method for the detection of both wild‐type (WT) and columnar (Co) alleles originating from ‘McIntosh Wijcik’ in a single reaction using a real‐time PCR allelic discrimination approach. Our assay was tested in 130 genotypes (80 normal and 50 columnar habit) from various crossings and their parents, and in all cases, genotype corresponded to phenotype. The assay is thus suitable for laboratories interested in high‐throughput MAS for columnar tree habit breeding.

Columnar growth phenotype in apple results from gibberellin deficiency by ectopic expression of a dioxygenase gene.

The apple cultivar McIntosh Wijcik, which is a mutant of 'McIntosh', exhibits a columnar growth phenotype (short internodes, few lateral branches, many spurs, etc.) that is controlled by a dominant Co gene. The candidate gene (MdDOX-Co), encoding a 2-oxoglutarate-dependent dioxygenase, is located adjacent to an insertion mutation. Non-columnar apples express MdDOX-Co in the roots, whereas columnar apples express MdDOX-Co in the aerial parts as well as in the roots. However, the function of MdDOX-Co remains unknown. Here, we characterized tobacco plants overexpressing MdDOX-Co. The tobacco plants showed the typical dwarf phenotype, which was restored by application of gibberellin A3 (GA3). Moreover, the dwarf tobacco plants had low concentrations of endogenous bioactive gibberellin A1 (GA1) and gibberellin A4 (GA4). Similarly, 'McIntosh Wijcik' contained low endogenous GA4 concentration and its dwarf traits (short main shoot and internodes) were partially reversed by GA3 application. These results indicate that MdDOX-Co is associated with bioactive GA deficiency. Interestingly, GA3 application to apple trees also resulted in an increased number of lateral branches and a decrease in flower bud number, indicating that gibberellin (GA) plays important roles in regulating apple tree architecture by affecting both lateral branch formation (vegetative growth) and flower bud formation (reproductive growth). We propose that a deficiency of bioactive GA by ectopic expression of MdDOX-Co in the aerial parts of columnar apples not only induces dwarf phenotypes but also inhibits lateral branch development and promotes flower bud formation, and assembly of these multiple phenotypes constructs the columnar tree form.

A root-localized gene in normal apples is ectopically expressed in aerial parts of columnar apples

The columnar apple ‘McIntosh Wijcik’, which is a mutation of ‘McIntosh’ shoots, has short internodes, thick stems, upright growth, poor lateral branches, and increased spur density. These columnar traits are controlled by a single dominant gene known as Co. We previously identified a putative dioxygenase gene (designated as 91071) as a promising Co candidate (expressed in the shoot apices of ‘McIntosh Wijcik’). However, tissue expression and function of the 91071 gene in noncolumnar apples is still not clear. In this study, we used reverse transcription polymerase chain reaction to demonstrate that the 91071 gene is mainly expressed in the roots of noncolumnar apples, whereas it is also expressed in shoot apices and leaves of columnar apples. In situ hybridization revealed that the 91071 gene is expressed at the primordium of lateral roots and root tips of both noncolumnar and columnar apple trees and in the shoot meristem and leaf primordium in the columnar apple ‘McIntosh Wijcik’. Grafting experiments of noncolumnar scion onto columnar rootstocks revealed that the columnar growth phenotype is not transmissible from rootstock to scion. These results indicated that ectopic expression of the 91071 gene in aerial parts causes columnar growth, whereas the expression of the 91071 gene in roots does not produce columnar growth. Furthermore, transgenic apples overexpressing the 91071 gene showed larger median adventitious root length and higher median number of lateral roots than control apples. Our result suggests that the 91071 gene may be related to root development.

Expression of a putative dioxygenase gene adjacent to an insertion mutation is involved in the short internodes of columnar apples (Malus × domestica).

Determining the molecular mechanism of fruit tree architecture is important for tree management and fruit production. An apple mutant 'McIntosh Wijcik', which was discovered as a bud mutation from 'McIntosh', exhibits a columnar growth phenotype that is controlled by a single dominant gene, Co. In this study, the mutation and the Co gene were analyzed. Fine mapping narrowed the Co region to a 101kb region. Sequence analysis of the Co region and the original wild-type co region identified an insertion mutation of an 8202bp long terminal repeat (LTR) retroposon in the Co region. Segregation analysis using a DNA marker based on the insertion polymorphism showed that the LTR retroposon was closely associated with the columnar growth phenotype. RNA-seq and RT-PCR analysis identified a promising Co candidate gene (91071-gene) within the Co region that is specifically expressed in 'McIntosh Wijcik' but not in 'McIntosh'. The 91071-gene was located approximately 16kb downstream of the insertion mutation and is predicted to encode a 2-oxoglutarate-dependent dioxygenase involved in an unknown reaction. Overexpression of the 91071-gene in transgenic tobaccos and apples resulted in phenotypes with short internodes, like columnar apples. These data suggested that the 8202bp retroposon insertion in 'McIntosh Wijcik' is associated with the short internodes of the columnar growth phenotype via upregulated expression of the adjacent 91071-gene. Furthermore, the DNA marker based on the insertion polymorphism could be useful for the marker-assisted selection of columnar apples.

Heritability of powdery mildew and scab resistance within apple progenies

Blažek J., Zelený L., Křelinova J. (2016): Heritability of powdery mildew and scab resistance within apple progenies. Plant Protect. Sci., 52: 123–127. The occurrence of both diseases was evaluated for four years in a selected orchard where 22 different apple progenies, totalling 699 seedlings were planted. During this period, no chemical protection against the diseases was applied in the orchard, and the incidence of the diseases was rated using a 9-point scale (from 9 = not infected to 1 = very highly infected). For the finalisation of the results, each seedling was characterised by the highest rate of infection from all four years of the evaluation. The lowest mean level of mildew infection equal to 7.47 was observed in the progeny of Resista × McIntosh Wijcik. Concerning the scab, the best was the progeny of Resista × Karmina having a rating equal to 8.27. The highest level of segregation of seedlings having joint resistance to both diseases was found out in the progenies obtained by crossings of HL665 × HL782 and Resista × HL447.

The columnar mutation (“Co gene”) of apple (Malus × domestica) is associated with an integration of a Gypsy-like retrotransposon

The columnar growth habit of apple trees (Malus × domestica Borkh.) is a unique plant architecture phenotype that arose as a bud sport mutation of a McIntosh tree in the 1960s. The mutation (“Co gene”) led to trees (McIntosh Wijcik) with thick, upright main stems and short internodes that generate short fruit spurs instead of long lateral branches. Although Co has been localized to chromosome 10, in a region approximately between 18.5 and 19 Mb, its molecular nature is unknown. In a classical positional cloning approach in combination with the analysis of NGS data, we cloned and analyzed the Co region. Our results show that the insertion of a Ty3/Gypsy retrotransposon into a non-coding region at position 18.8 Mb is the only detectable genomic difference between McIntosh and McIntosh Wijcik and is found in all columnar cultivars. The genetic effect of the insertion is unclear; however, Illumina® RNA-seq data of McIntosh and McIntosh Wijcik suggest that the columnar growth habit is associated with differential expression of the retrotransposon transcript, causing changes in the expression levels of many protein coding genes. The mechanism by which the Gypsy retrotransposon is involved in generating the columnar habit is not yet clear; our findings form the basis for tackling this question.

HYBRIDIZING 'MCINTOSH WIJCIK' AND HEAT-TOLERANT APPLE CULTIVARS TO DEVELOP PRECOCIOUS SEEDLING TREES WITH IMPROVED TREE ARCHITECTURE

ISHS IX International Symposium on Integrating Canopy, Rootstock and Environmental Physiology in Orchard Systems HYBRIDIZING 'MCINTOSH WIJCIK' AND HEAT-TOLERANT APPLE CULTIVARS TO DEVELOP PRECOCIOUS SEEDLING TREES WITH IMPROVED TREE ARCHITECTURE

Tree growth and some other characteristics of new columnar apple cultivars bred in Holovousy, Czech Republic

Several characteristics related to tree growth, their fruitage and resistance against scab and powdery mildew were assessed during 2007–2008 on five new cultivars in comparison with the ancestral McIntosh Wijcik, in which 4 out of 5 had their pedigree. In several plots established between 1998 and 2006, the trees, which were on different rootstocks predominantly planted in spacing 4 × 0.5 m, were grown as vertical cordons practically without any pruning or shaping. Besides the first years after the planting these columnar trees generated very limited side branching. The cultivars mutually differ in tree vigour, spurring density, and size of fruiting spurs. Mean annual prolongation of central axes in the case of the trees on vigorous rootstocks varied within 30.8–57.5 cm, however, on M 9 rootstock it was distinctly shorter (23.4–34.1 cm). Beyond cultivar and rootstock the length of the annual prolongation was also significantly influenced by fruit set level. Trees of the Kordona cultivar were generally the most vigorous while those of Pidi were the weakest. The biennial pattern of bearing observed to an extent on nearly all the cultivars was connected with tendency to over-crop the trees. The highest annual yield equal to 10.3 kg was generated on trees of Kordona on MM 106, which corresponds to a total harvest of 25.7 t/ha. Cultivars Herald and Slendera possessed combined resistance both to scab and powdery mildew, whereas Kordona and Cumulus were resistant only to scab, but were in different degrees susceptible to mildew.

Overexpression of LEAFY in apple leads to a columnar phenotype with shorter internodes

To break the juvenile stage of apple (Malus x domestica Borkh.) we transferred the LFY gene of Arabidopsis into the genome of the apple cv. 'Pinova'. A total of five transgenic clones constitutively overexpressing the LFY gene were obtained. Approximately, 20 shoots of each clone were rooted and transferred to the glasshouse. No flowers were obtained on transgenic plants during the first 2 years of cultivation. Evaluation of the expression of possible LFY targets revealed that no transcripts could be detected for MdAP1-1 and MdAP1-2. MdTFL1 was unaffected. Based on the absence of the LFY core-binding sequence within promoter sequences of MdAP1-1 and MdAP1-2, it was concluded that LFY was not able to induce these genes. The LFY genes of apple were unaffected in transgenic plants and sequence alignments of the C-terminal amino acid sequence showed a high conservation of these proteins. A change in binding ability to DNA can therefore be excluded. Instead of early flowering, the transgenic plants showed an altered phenotype, which is similar to the columnar phenotype of the 'McIntosh Wijcik' mutant of apple. The transgenic plants showed shortened internodes and a significantly reduced length of the regrowing shoot. A negative correlation was observed between the length of the regrowing shoot and the LFY mRNA transcript level. Furthermore, the LFY transgenic apple plants showed an increased shoot diameter at node 20, which was positively correlated with the LFY mRNA transcript level. Based on our results, we assume an alternative role of LFY in apple.

Plant Regeneration from Leaf and Stem Segments of Shoots Raised in vitro from Mature Apple Trees

Summary Plants were regenerated from leaf segments of shoots raised in vitro from mature apple trees of the cultivars Akero, McIntosh, McIntosh Wijcik, Gravenstein and the apple rootstock M26. The regeneration capacity was influenced by wounding, macroelements, light conditions, polarity and maturity of the leaves. The highest number of shoots per leaf segment was formed using young expanding leaves on a medium based on N6 macroelements plus 22 µM BAP and 1 µM NAA. Dark treatment during the first 3 weeks of culture and placing the leaf segments on the medium with the abaxial surface uppermost enhanced shoot formation. The morphogenetic response for stem segments was very low since only McIntosh Wijcik formed shoots and at a low frequency. The histological studies revealed both embryoidlike structures and adventitious shoots. Shoots regenerated from both leaves and meristems were rooted and placed in the greenhouse for comparison. Trees of the cultivars McIntosh, McIntosh Wijcik and M26 were planted in the field for further evaluation of growth habit and fruit setting.

Relationship of Endogenous Gibberellin and Cytokinin Levels in Shoot Tips to Apical Form in Four Strains of ‘McIntosh’ Apple

Abstract Shoot tips collected in June from ‘Summerland Red McIntosh’ (nonspur), ‘Macspur’, ‘Morspur’, and ‘McIntosh Wijcik’ apple (Malus domestica Borkh.) were examined for polar and less-polar gibberellins (GAs) and for cytokinin-like bioactivity. The latter three strains are increasingly compact and spurry, with ‘McIntosh Wijcik’ exhibiting a distinctive columnar growth habit. ‘McIntosh Wijcik’ shoot tips exhibited significantly lower levels of polar GAs, but the levels of less-polar GAs were similar in all four strains. Conversely, ‘McIntosh Wijcik’ displayed higher levels of cytokinin-like substances than the other three strains. These results are discussed in relation to the physiology of the spur-type growth habit of ‘McIntosh Wijcik’ apple and its progeny.

ABSCISIC ACID LEVELS AND GENETIC COMPACTION IN APPLE SEEDLINGS

Apple (Malus sylvestris Mill.) seedlings arising from controlled crosses involving McIntosh Wijcik segregate reliably into normal and compact growth types. The compact or spur-type seedlings have shorter, thicker stems; shorter internodes; and a strong upright, nonbranching growth habit. They contained less free ABA per shoot tip than normal seedlings. A reliable and rapid method for extracting and measuring ABA is described.

Inheritance of Compact Growth Type in Apple1

Abstract Test crosses within the progeny of ‘Golden Delicious’ × ‘McIntosh Wijcik’ confirmed the hypothesis that the typical growth habit of ‘McIntosh Wijcik’ is determined by a single dominant gene, Co. A small but consistent deficiency in the compact class suggested the presence of modifier gene or genes. Two independently originated natural mutants, ‘McIntosh Wijcik’ and ‘McIntosh Bendig’, were considered identical. The compact-growth seedlings, particularly the homozygous dominant genotypes from backcrosses and sib-crosses, constitute valuable breeding material. Compact segregates, evaluated as mature trees, were deemed to be suitable for very dense plantings.

Spur Type Growth Habit in 60 Apple Progenies1,2

Abstract Growth habit at bearing age was studied in 60 progenies of apple, Malus pumila Mill. One progeny, ‘Golden Delicious’ × ‘McIntosh Wijcik’, segregated into 2 distinct growth types, spur-type (compact) and non-spur (standard) at an approximate 1:1 ratio. In the other 59 progenies there was a continuous variation from trees with no or few spurs to trees with many spurs. Trees were classified into 5 arbitrary “spurriness” classes, mainly based on the ratio of spurs to vegetative shoots. Average spurriness of a progeny generally increased with that of its parents. Spur-type mutants of standard cultivars when used as both parents increased the degree of spurriness slightly, but less than expected. Certain progenies had 30 to 56% trees comparable in spurriness to the spur-types of commercial apple cultivars. Among parents inducing spurriness in the progeny, ‘Melba’ was outstanding.