Markers and Genetics The ability to use molecular markers for

pre-selection at the seedling stage, prior to field planting, is of great importance. The ability to prescreen reduces land and greenhouse requirements, saves money and results in a higher percentage of desirable seedlings being planted in the field.

3.7.1 Isozymes

Isozymes were the first markers to be used in apple breeding. They have been used for identification of cultivars, for estimating genetic diversity in germplasm collections and to confirm or refute suspected parent-age. Chyi and Weeden (1984) used isozymes to determine that the female parent supplied the unreduced diploid gamete in several triploid cultivars. Weeden and Lamb (1987) studied isozyme polymorphism in nine enzyme systems and identified four linkage groups. Close linkage of glutamate oxaloac-etate transaminase (Got-1) and the S incom-patibility locus was reported (Manganaris and Alston, 1987). Manganaris and Alston (1988) also found that the acid phosphatase gene ACP-1 was linked with the endopepti-dase gene ENP-1 and the pale green lethal gene, l. In 1994, Manganaris et al. reported that the isozyme locus Pgm-1 was tightly linked to V_f scab resistance. Advances con-tinue to be made. Alston et al. (2000) reviewed research on 69 isozymes in apple.

3.7.2 Scab resistance

The amount of research focused on the iden-tification and development of markers for the V_fgene is extensive. Only the most recent studies will be discussed. Patocchi et al.

(1999) used markers to fine-map the V_f region. Xu and Korban (2000) used AFLP markers for saturation mapping of V_f and then sequence-characterized amplified regions (SCARs) were developed from these AFLP markers (Xu et al., 2001). These are prerequisites for map-based cloning.

Markers have also been developed for the V_mregion from M. atrosanguinea 804 (Cheng et al., 1998). Research on markers for the other genes for scab resistance is progressing in several laboratories.

3.7.3 Powdery mildew

Molecular markers for the resistance genes Pl₁from M.× robusta (Markussen et al., 1995), Pl₂from M.× zumi Rehd. (Dunemann et al.,

1999) and Pl_wfrom ‘White Angel’ (Batlle and Alston, 1996) are being used in marker-assisted selection.

3.7.4 Insect resistance

Markers have been developed for resistance to the rosy leaf-curling aphid (Dysaphis devecta Wlk.) (Roche et al., 1997a,b) and to woolly apple aphid (Eriosoma lanigerum Hausmn.), an important pest in breeding rootstocks (Bus et al., 2000).

3.7.5 Incompatibility alleles

The self-incompatibility system in apple is well known, but until recently the number of S alleles involved and the extent of cross-incompatibility was not known. The cloning and molecular analysis of two self-incom-patibility alleles from apple (Broothaerts et al., 1995) was followed by the development of a molecular method for S-allele identifica-tion in apple based on allele-specific PCR (Janssens et al., 1995). The alleles S2, S3, S5, S7 and S9 were identified and used to geno-type several cultivars, including ‘Idared’

(S3S7), ‘Fiesta’ (S3S5), ‘Jonathan’ (S7S9),

‘Elstar’ (S3S5), ‘Gala’ (S2S5) and ‘Golden Delicious’ (S2S3). Sakurai et al. (1997, 2000) used this method to genotype Japanese and American apple cultivars and advanced selections. Six additional S alleles were sequenced: S4, S24, S26, S27, Sd and Sf (Sassa et al., 1996; Katoh et al., 1997;

Verdoodt et al., 1998). S genotyping has raised questions of paternity in that several cultivars have S alleles different from those predicted by their parentage.

S-allele genotyping has also been used to assess homozygosity in shoots obtained through haploid induction by screening in vitro shoots for single S alleles as opposed to S alleles of a parent whose pollen was irradi-ated and used to stimulate parthenogenic development (Verdoodt et al., 1998). Van Nerum et al. (2000) transformed ‘Elstar’ with an S allele in the sense or antisense direc-tion and analysed lines for self-fertility.

Fluorescent microscopy confirmed that some

lines appeared to have the incompatibility mechanism switched off, as evidenced by pollen-tube growth.

3.7.6 Other traits

Markers have been identified for many traits, including fruit skin colour (Cheng et al., 1996), columnar habit (Hemmat et al., 1997) and fruit acidity (malic acid concentration) (Conner et al., 1997). Conner et al. (1998) used RAPDs to estimate the position and effect of QTL affecting juvenile tree growth and development and found that a large number of traits had significant variation associated with the map position of the dominant columnar gene, Co.

3.7.7 Microsatellites/simple sequence repeats (SSRs)

SSRs, short tandem repeats of one to six base pairs, have been used in cultivar identifica-tion and genetic analysis and to reveal iden-tities, genetic diversity and relationships in a core subset collection. Guilford et al. (1997) used SSRs in a survey of 21 cultivars. The majority of SSRs were highly polymorphic and diploid and showed simple Mendelian inheritance, although about 25% of markers generated complex banding patterns. Three microsatellite markers were sufficient to dif-ferentiate between all 21 cultivars.

Gianfranceschi et al. (1998) developed 16 SSR markers that amplified all alleles from 19 cultivars, breeding selections and M. flori-bunda 821. Two selected SSRs were able to distinguish all cultivars except ‘Starking’ and

‘Red Delicious’. Hokanson et al. (1998) screened accessions from a core subset of the germplasm repository with eight SSRs. The primer pairs differentiated all but seven pairs of accessions.

3.7.8 Molecular maps

The first linkage map of apple was based on a progeny of 56 seedlings from a cross of

‘Rome Beauty’ × ‘White Angel’ that

com-bined isozyme, RAPD and restriction-fragment length polymorphism (RFLP) markers (Hemmat et al., 1994). Conner et al.

(1997) developed maps for ‘Wijcik McIntosh’ and for two advanced scab-resis-tant selections from the Cornell breeding programme. Maliepaard et al. (1999) reviewed the maps for ‘Prima’ × ‘Fiesta’, the first progeny used for mapping apple in Europe. One hundred and fifty-five F₁ seedlings were genotyped with 208 mark-ers, which included RFLPs, RAPDS, isoen-zymes and microsatellites. The European DARE programme has as one of its goals the identification of molecular markers linked to genes for resistance. Five molecu-lar maps, mainly involving SSRs and AFLPs, have been constructed. Research is conducted in France, Germany, Italy, Greece, The Netherlands and Switzerland.

Mapping populations also include ‘Fiesta’

× ‘Discovery’; ‘Fiesta’ is susceptible and

‘Discovery’ has a high level of resistance to both scab and mildew. Researchers at INRA in France are using the cross ‘Discovery’ × TN10-8. Researchers at the University of Bologna in Italy and in Greece are mapping

‘Durello di Forli’ × ‘Fiesta’, while those in Ahrensberg, Germany, are mapping ‘Prima’

× ‘Discovery’. A common set of AFLPs will be tested in mapping populations that have

‘Discovery’ as a parent.

3.7.9 Comparative mapping

Mapping of resistance-gene analogues (RGAs) from other species is ongoing.

Comparative mapping with other members of the Rosaceae, especially with Pyrus and Prunus, is likely. Several microsatellite repeats in peach (Prunus persica (L.) Batsch.) were also amplified in apple (Cipriani et al., 1999).

3.7.10 Bacterial artificial chromosome (BAC) library of apple

Vinatzer et al. (1998) reported the construc-tion of a BAC library using ‘Florina’, a scab-resistant cultivar (V_fgene). The BAC library

is a prerequisite for the construction of a physical map of apple and for map-based cloning of V_for other apple genes.

3.8 Biotechnology

Name recognition in marketing apples is important. Thus, using biotechnology to change a key characteristic of a popular com-mercial cultivar and yet maintain varietal identity is very desirable. This objective can-not be achieved in traditional breeding because of the need to use a modified back-cross procedure due to self-incompatibility and inbreeding depression.

3.8.1 Somaclonal variation

There has been an ongoing debate about the effect of tissue culture on apple and the extent of somaclonal variation that might exist. This has important implications for the genetic transformation and regeneration of commercial cultivars. Somaclonal varia-tion for resistance to the fire blight pathogen E. amylovora and for alterations to rooting ability and shoot proliferation in vitro was examined by Donovan et al.

(1994a,b). Zimmerman (1997) reported that micropropagated trees of ‘Redspur Delicious’ exhibited tree-to-tree variation and that most replicates did not maintain the spur habit. However, micropropagating spur-type trees from previously microprop-agated trees that did retain the spur habit was successful in having spur habit main-tained. When tissue culture-derived ‘Gala’

and ‘Royal Gala’ clones that were obtained via axillary and adventitious bud formation were compared with conventionally grafted trees by McMeans et al. (1998), very little somaclonal variation was observed in mor-phological or reproductive traits (Plate 3.5).

Yet Zimmerman and Steffens (1995) reported that tissue-cultured ‘Gala’ trees often developed burr knots 6–7 years after being transferred to the field. They sug-gested that tissue cultures should be re-established annually to prevent this problem.

3.8.2 Regeneration and transformation The literature in this area is extensive. The reader is referred to several reviews as a starting-point, but advances are continually being made. Protoplast fusion (symmetric and asymmetric) has been tested in apple and some tentative somatic hybrids have been identified (Huancaruna Perales et al., 2000). Singh and Sansavini (1998) reviewed transformation across fruit crops, while Hammerschlag (2000) reviewed transforma-tion of Malus. De Bondt et al. (1994, 1996) reviewed factors influencing gene-transfer efficiency during early transformation steps and factors affecting regeneration of trans-formants. Maximova et al. (1998) investi-gated transformation using green fluorescent protein and found that high transient expres-sion and low stable transformation sug-gested that factors other than (T)-DNA transfer were rate-limiting.

3.8.3 Transgenes introduced The first report of transformation of apple occurred in 1989 (James et al., 1989).

Trifonova et al. (1994) transformed ‘Granny Smith’ with nptII and ipt genes, encoding for one of the first enzymes in the cytokinin biosynthetic pathway (Fig. 3.1). In 1995, Yao et al. introduced the acetolactate synthase gene into ‘Royal Gala’ to increase resistance

to the herbicide Glean™ in transgenic plants.

James et al. (1996) documented the stable expression and Mendelian segregation of the marker transgenes nopaline synthase (nos) and the cotransferred gene neomycin phos-photransferase (nptII) in the flesh of apple fruits 7 years after the initial transformation.

In 1996, ‘Gala’, ‘Golden Delicious’ and

‘Elstar’ were transformed (Puite and Schaart, 1996), followed by ‘Delicious’ and ‘Pink Lady^®’ (Sriskandarajah and Goodwin, 1998) and ‘Delicious’, ‘Greensleeves’ and ‘Royal Gala’ (Maximova et al., 1998).

Bolar et al. (1999) developed an efficient transformation system for ‘Marshall McIntosh’. Expression of endochitinase from Trichoderma harzianum in apple increased resistance to scab and reduced vigour in transgenic ‘Marshall McIntosh’ (Bolar et al., 2000). There was a significant negative corre-lation between the level of endochitinase production and both the amount of disease and plant growth.

Yao et al. (1999a) grew transgenic ‘Royal Gala’ apple trees under controlled green-house conditions and 20% of the trees flow-ered in the second year, but, when scion wood from the top of these clones was grafted on to M.9, 85% produced flowers and fruit the next year. Inheritance of three trans-genes, uid A, neomycin phototransferase II and acetolactate, fit a 1 : 1 ratio in most lines, but in one progeny line the T-DNA integra-tion pattern was complex.

Fig. 3.1. Machine used to transform apple tissue by inserting genes.

Two of four transgenic lines possessing the kanamycin resistance gene and antisense polyphenol oxidase (PPO) DNA showed repressed PPO activity and a lower brown-ing potential than control shoots (Murata et al., 2000). Broothaerts et al. (2000b) developed a spectrophotometric assay for the analysis of PPO in apple and tobacco leaves to increase efficiency in screening large num-bers of transgenic plants (Fig. 3.2).

Transformation of ‘Jonagold’ with antimi-crobial peptide genes (A1-AMP) resulted in 28 independent transgenic lines, which are being tested for resistance to apple scab using artificial inoculation assays (Broothaerts et al., 2000a). At the Apple Research Centre in Morioka, Japan, ‘Orin’

and the Japanese rootstock ‘JM 7’ have been transformed with genes encoding the sor-bitol-metabolizing enzyme sorbitol-6-phosphate dehydrogenase isolated from apple, chitinase isolated from rice, glucanase from soybean and sacrotoxin from the flesh-fly (Soejima et al., 2000).

3.8.4 Rootstocks transformed Apple rootstocks are also a focus in biotech-nology, with M.26 rootstock (Lambert and Tepfer, 1992; Maheswaran et al., 1992;

Holefors et al., 1998) and M.7 rootstock trans-formed (Norelli et al., 1999). M.26 was also transformed with rolA and rolB (Zhu and Welander, 2000). Zhu et al. (2001) trans-formed M.9 rootstock with rolB and found that in in vitro rooting tests all transgenic

clones rooted (83–100%) on hormone-free rooting medium versus 1% for the controls.

Root length and root morphology did not differ between transgenic clones and the untransformed controls.

3.8.5 Transgene silencing

Ko et al. (1998) found that there were alter-ations in nptII and gus expression following micropropagation of transgenic M.7 apple rootstock lines. The gus gene was present in non-staining lines. Gus gene silencing was due to methylation in some cases, but in others the mixed staining might be due to a mixture of transformed and non-transformed cells.

3.8.6 Challenges

At present, traits that are complex (e.g. yield and flavour) are not likely candidates for improvement by biotechnology. Additionally, there is a need for genes from Malus to be cloned, since public concern about transgene technology does differentiate between native and non-native genes. There is also a need for specific promoters, wound-inducible or fruit- or leaf-specific, so that gene expression may be targeted only to the parts of the plant necessary for the desired effect (Gittins et al., 2000). Transgenic testing must ensure that there are no non-target effects and that transgenic lines are stable and non-chimeric.

Fig. 3.2. Gel comparing banding patterns of seedlings segregation for disease resistance.

3.9 Future Prospects