Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.)

Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene.     

Characterization of the flanking regions of the S-RNase genes of Japanese pear (Pyrus serotina) and apple (Malus×domestica)

Genomic sequences of the self-incompatibility genes, the S-RNase genes, from two rosaceous species, Japanese pear and apple, were characterized. Genomic Southern blot and sequencing of a 4.5-kb genomic clone showed that the S⁴-RNase gene of Japanese pear is surrounded by repetitive sequences as in the case of the S-RNase genes of solanaceous species. The flanking regions of the S²- and S^f-RNase genes of apple were also cloned and sequenced. The 5′ flanking regions of the three alleles bore no similarity with those of the solanaceous S-RNase genes, although the position and sequence of the putative TATA box were conserved. The putative promoter regions of the Japanese pear S⁴- and apple S^f-RNase genes shared a stretch of about 200 bp with 80% sequence identity. However, this sequence was not present in the S²-RNase gene of apple, and thus it may reflect a close relationship between the S⁴- and S^f-RNase genes rather than a cis-element important in regulating gene expression. Despite the uniform pattern of expression of the rosaceous S-RNase genes, sequence motifs conserved in the 5′ flanking regions of the three alleles were not found, implying that the cis-element controlling pistil specific gene expression also locates at the intragenic region or upstream of the analyzed promoter region.     

Molecular characterization of newS-RNases (‘S31’ and ‘S32’) in apple (Malus ×domestica Borkh.)’) in apple (Malus ×domestica Borkh.)

Apple exhibits gametophytic self-incompatibility (GSI) that is controlled by the multiallelic S-locus. This S-locus encodes polymorphicS ribonuclease (S-RNase) for the pistil-part 5 determinant. Information aboutS-genotypes is important when selecting pollen donors for fruit production and breeding of new cultivars. We determined the 5-genotypes of ‘Charden’ (S₂S₃S₄), ‘Winesap’ (S₁S₂₈), ‘York Imperial’ (S₂S₃₁), ‘Stark Earliblaze’ (S₁S₂₈), and ‘Burgundy’ (S₂₀S₃₂), byS-RNase sequencing and S-allele-specific PCR analysis. Two newS-RNases, S₃₁ and S₃₂, were also identified from ‘York Imperial’ and ‘Burgundy’, respectively. These newS-alleles contained the conserved eight cysteine residues and two histidine residues essential for RNase activity. Whereas S₃₁ showed high similarity to S₂₀ (94%), S₃₂ exhibited 58% (to S₂₄) to 76% (to S₂₅) similarity in the exon regions. We designed newS-allele-specific primers for amplifying S₃₁- and S₃₂-RNasc-specific fragments; these can serve as specific gene markers. We also rearranged the apple S-allele numbers containing those newS-RNases. They should be useful, along with anS-RNase-based PCR system, in determining S-genotypes and analyzing new alleles from apple cultivars.     

Creating a saturated reference map for the apple (Malus × domestica Borkh.) genome

The availability of a high quality linkage map is essential for the detection and the analysis of quantitative traits. Such a map should cover a significant part of the genome, should be densely populated with markers, and in order to gain the maximum advantage should be transferable to populations or cultivars other than the ones on which it has been constructed. An apple genetic linkage map has been constructed on the basis of a segregating population of the cross between the cultivars Fiesta and Discovery. A total of 840 molecular markers, 475 AFLPs, 235 RAPDs, 129 SSRs and 1 SCAR, were used for the two parental maps constructed with JoinMap and spanning 1,140 cM and 1,450 cM, respectively. Large numbers of codominant markers, like SSRs, enable a rapid transfer of the map to other populations or cultivars, allowing the investigation of any chosen trait in another genetic background. This map is currently the most advanced linkage map in apple with regard to genome coverage and marker density. It represents an ideal starting point for future mapping projects in Malus since the stable and transferable SSR frame of the map can be saturated quickly with dominant AFLP markers.     

Use of a feedback-insensitive α subunit of anthranilate synthase as a selectable marker for transformation of rice and potato

A selection system based on a mutant rice gene for a feedback-insensitive subunit of anthranilate synthase (OASA1D) was developed for the transformation of rice and potato. Expression of OASA1D conferred resistance to the tryptophan analog 5-methyltryptophan (5MT) in transformed cells of rice and potato. The selection system based on OASA1D and 5MT was associated with a high transformation efficiency, a short time frame for the generation of transgenic plants, simple culture procedures, and it was as effective as hygromycin B selection in rice (monocotyledon) and kanamycin selection in potato (dicotyledon). Transgenic rice and potato plants established by 5MT selection had normal morphology and accumulated tryptophan when OASA1D was expressed under the control of a constitutive promoter. These results demonstrate the efficacy of OASA1D as a selectable marker and they suggest that the 5MT selection system based on this gene will prove applicable to a wide range of plant species and culture procedures.     

Common and specific responses to iron and phosphorus deficiencies in roots of apple tree (Malus × domestica)

Plant Molecular Biology - Iron and phosphorus are abundant elements in soils but poorly available for plant nutrition. The availability of these two nutrients represents a major constraint for...     

Application of a high-speed breeding technology to apple (Malus × domestica) based on transgenic early flowering plants and marker-assisted selection

Breeding of apple (Malus × domestica) remains a slow process because of protracted generation cycles. Shortening the juvenile phase to achieve the introgression of traits from wild species into prebreeding material within a reasonable time frame is a great challenge. In this study, we evaluated early flowering transgenic apple lines overexpressing the BpMADS4 gene of silver birch with regard to tree morphology in glasshouse conditions. Based on the results obtained, line T1190 was selected for further analysis and application to fast breeding. The DNA sequences flanking the T-DNA were isolated and the T-DNA integration site was mapped on linkage group 4. The inheritance and correctness of the T-DNA integration were confirmed after meiosis. A crossbred breeding programme was initiated by crossing T1190 with the fire blight-resistant wild species Malus fusca. Transgenic early flowering F(1) seedlings were selected and backcrossed with 'Regia' and 98/6-10 in order to introgress the apple scab Rvi2, Rvi4 and powdery mildew Pl-1, Pl-2 resistance genes and the fire blight resistance quantitative trait locus FB-F7 present in 'Regia'. Three transgenic BC'1 seedlings pyramiding Rvi2, Rvi4 and FB-F7, as well as three other BC'1 seedlings combining Pl-1 and Pl-2, were identified. Thus, the first transgenic early flowering-based apple breeding programme combined with marker-assisted selection was established.     

Cryopreservation of apple (Malus×domestica Borkh.) shoot tips following encapsulation-dehydration or encapsulation-vitrification

Axillary shoot tips of apple cv. Golden Delicious isolated from shoot cultures were successfully cryopreserved using the encapsulation-dehydration technique. After encapsulation in alginate gel, embedded shoot tips were dehydrated by exposure to a sterile air flow before being frozen in liquid nitrogen and subsequent slow thawing. A preculture on modified MS medium containing 0.75 M sucrose followed by 6 h of dehydration (21% residual water) led to the highest shoot regrowth of frozen, coated shoot tips (83.7%). Among the sugars tested, sucrose and sorbitol presented the best cryoprotective effect. Four other scion apple varieties and rootstocks were also successfully cryopreserved. Axillary shoot tips of five apple (Malus×domestica Borkh.) scion and rootstock cultivars were cryopreserved using the encapsulation-vitrification technique. Using a one-step freezing method, we successfully cryopreserved axillary shoot tips without the requirement of a cold hardening pretreatment of the shoot cultures. Cryopreserved shoot tips treated with aqueous cryoprotective mixture IV containing 180% (w/v) sucrose and 120% (v/v) ethylene glycol showed the highest shoot regrowth rates, which varied from 64% to 77%, depending on the cultivar. Received: 29 July 1999 / Revision received: 24 September 1999 / Accepted: 26 November 1999     

Genetic and physical characterisation of the locus controlling columnar habit in apple (Malus × domestica Borkh.)

A better understanding of the genetic control of tree architecture would potentially allow improved tailoring of newly bred apple cultivars in terms of field management aspects, such as planting density, pruning, pest control and disease protection. It would also have an indirect impact on yield and fruit quality. The Columnar (Co) locus strongly suppresses lateral branch elongation and is the most important genetic locus influencing tree architecture in apple. Co has previously been mapped on apple linkage group (LG) 10. In order to obtain fine mapping of Co, both genetically and physically, we have phenotypically analysed and screened three adult segregating experimental populations, with a total of 301 F₁ plants, and one substantial 3-year old population of 1,250 F₁ plants with newly developed simple sequence repeat (SSR) markers, based on the ‘Golden delicious’ apple genome sequence now available. Co was found to co-segregate with SSR marker Co04R12 and was confined in a region of 0.56 cM between SSR markers Co04R11 and Co04R13, corresponding to 393 kb on the ‘Golden delicious’ genome sequence. In this region, 36 genes were predicted, including at least seven sequences potentially belonging to genes that could be considered candidates for involvement in control of shoot development. Our results provide highly reliable, virtually co-segregating markers that will facilitate apple breeding aimed at modifications of the tree habit and lay the foundations for the cloning of Co.     

One-step reconstruction of multi-generation pedigree networks in apple (Malus × domestica Borkh.) and the parentage of Golden Delicious

The increasing availability of genomic tools improves our ability to investigate the patterns of genetic diversity and relatedness among individuals. The pedigrees of many apple cultivars are completely unknown, often reducing the efficiency of breeding programs. Using a multilocus simple sequence repeat dataset, we applied a novel multi-generation pedigree-network reconstruction procedure based on the software FRANz in a Malus × domestica collection (101 cultivated and 22 wild apples) with partially known pedigree relationships. The procedure produced 78 parent–offspring relationships organized into three networks and showed high power for detecting real pedigree links (98.5 %) and a low false-positive rate (9.0 %). The largest reconstructed pedigree network spanned four generations and involved 65 cultivars. The availability of detailed pedigree connections confirmed that recent genealogical relationships affect population genetic structure in apple. Finally, our analysis enabled us to confirm or discard several pedigrees known only anecdotically, among which the cultivar Grimes Golden was validated as a parent of the widely grown cultivar Golden Delicious. The pedigree reconstruction protocol here described will be of broad applicability to other collections and crop species.     

Determination of partial genomic sequences and development of a CAPS system of the S-RNase gene for the identification of 22 S haplotypes of apple (Malus × domestica Borkh.)

Information about self-incompatibility (S) genotypes of apple cultivars is important for the selection of pollen donors for fruit production and breeding. Although S genotyping systems using S haplotype-specific PCR of S-RNase, the pistil S gene, are useful, they are sometimes associated with false-positive/negative problems and are unable to identify new S haplotypes. The CAPS (cleaved amplified polymorphic sequences) system is expected to overcome these problems, however, the genomic sequences needed to establish this system are not available for many S-RNases. Here, we determined partial genomic sequences of eight S-RNases, and used the information to design new primer and to select 17 restriction enzymes for the discrimination of 22 S-RNases by CAPS. Using the system, the S genotypes of three cultivars were determined. The genomic sequence-based CAPS system would be useful for S genotyping and analyzing new S haplotypes of apple.