Genetic characterization of the Ma locus with pH and titratable acidity in apple

Apple fruit flavor is greatly affected by the level of malic acid, which is the major organic acid in mature apple fruit. To understand the genetic and molecular basis of apple fruit acidity, fruit juice pH and/or titratable acidity (TA) were measured in two half-sib populations GMAL 4595 [Royal Gala?×?PI (Plant Introduction) 613988] and GMAL 4590 (Royal Gala?×?PI 613971) of 438 trees in total. The maternal parent Royal Gala is a commercial variety and the paternal parents are two M. sieversii (the progenitor species of domestic apple) elite accessions. The low-acid trait segregates recessively and the overall acidity variations in the two populations were primarily controlled by the Ma (malic acid) locus, a major gene discovered in the 1950s (Nybom in Hereditas 45:332?C350, 1959) and later mapped to linkage group 16 (Maliepaard et al. in Theor Appl Genet 97:60?C73, 1998). The allele Ma has a strong additive effect in increasing fruit acidity and is incompletely dominant over ma. QTL (quantitative trait locus) analyses in GMAL 4595 mapped the major QTL Ma in both Royal Gala and PI 613988, the effects of which explained 17.0?C42.3% of the variation in fruit pH and TA. In addition, two minor QTL, tentatively designated M2 and M3, were also detected for fruit acidity, with M2 on linkage group 6 of Royal Gala and M3 on linkage group 1 of PI 613988. By exploring the genome sequences of apple, eight new simple sequence repeat markers tightly linked to Ma were developed, leading to construction of a fine genetic map of the Ma locus that defines it to a physical region no larger than 150?kb in the Golden Delicious genome.     

Impact of European red mite on Golden Delicious and Oregon Spur apples in Israel

To develop a damage threshold for the European red mite (ERM), Panonychus ulmi (Koch), the effect of ERM injury on fruit weight and return crop in the apple cultivars (CVs) Golden Delicious and Oregon Spur were studied in the Upper Galilee of Israel for two consecutive seasons. The ERM significantly affected the leaf colour, chlorophyll content, fruit weight and return harvest in both CVs. High population levels of 450 adult female cumulative mite days (ACMDs) reduced the fruit weight on Golden Delicious but not on Oregon Spur. Population levels above approximately 600 and 900 ACMDs in Golden Delicious and Oregon Spur, respectively, caused a substantial reduction in the return harvest. Because no damage was observed at low population levels, an action threshold of 150 ACMDs is recommended.     

Characterisation of the virescent locus controlling a recessive phenotype in apple rootstocks (Malus pumila Mill.)

Certain progenies of Malling apple rootstocks (Malus pumila) have been reported to segregate for a virescent trait: leaves are chlorotic at germination or bud break but turn green as the season progresses. The M432 rootstock mapping progeny, from which a linkage map has recently been elaborated with 323 simple sequence repeat (SSR) markers and 3,069 single nucleotide polymorphism (SNP) markers, also segregates for this phenotype. In this investigation, 188 seedlings were scored and, on the basis of a 3:1 segregation, virescence was attributed to the recessive gene (vir) for which the two parents, M.27 and M.116, are heterozygous. At least seven of 28 Malling rootstocks are heterozygous for this apparently deleterious trait. With the published marker data the gene was mapped to linkage group 12, tightly flanked by the SSR CH01g12 and the SNP marker 475880474, and was located in a physical interval of 2.36 Mb on the Golden Delicious genome sequence. A PCR-based marker was developed from the SNP and along with the SSR was scored in a set of Malus rootstock accessions. The screening of this collection demonstrated that those accessions known to be heterozygous at the vir locus all carried the 152 allele of the SSR and the G allele of the SNP, whilst a virescent accession was homozygous for the alleles. The results we present here could help predict the genotype of apple rootstocks at the vir locus, assist in the fine mapping of the vir locus to identify potential candidate genes for the trait and also aid rootstock breeding.     

A test of fruit varieties on entry rate and development by neonate larvae of the codling moth,Cydia pomonella

The rate of entry by neonate larvae of the frugivorous codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), into fruit material was investigated. We used no‐choice bioassays in climate‐controlled rooms to assay larval entry across four host plant species (apple, pear, quince, walnut) and three varieties within a single fruit species (apple). Larvae successfully entering apples were reared to adulthood, and we collected tissue samples from apples which were successfully colonized in order to determine sucrose concentrations. This information was used to evaluate differences in adult moth size, development time, and pulp sucrose concentration due to apple variety. Four important findings emerged: (1) neonate larvae had the highest frequency of entry (86% of larvae) into apple fruits, compared with pear (78%), quince (56%), and walnut (32%); (2) the frequency of larval entry into immature apples differed across apple varieties, and larval entry rate was highest in variety Golden Delicious (72%), compared with Granny Smith (46%) and Red Delicious (64%); (3) on average, adult moths were larger and development times were shorter on the variety with the highest entry frequency (Golden Delicious); and (4) apple pulp sucrose concentrations were higher for Golden Delicious (17.5 μg mg^?1) than for either Granny Smith (15.9 μg mg^?1) or Red Delicious (15.1 μg mg^?1) varieties, which correlates positively with entry and development data. We conclude that host fruit species and varietals within a species affect the entry rate of neonate codling moth larvae in no‐choice assays. We hypothesize that larval development is influenced by mean sucrose concentrations or other phytochemical differences associated with host fruit varieties.     

A Multidisciplinary Approach Providing New Insight into Fruit Flesh Browning Physiology in Apple (Malus x domestica Borkh.)

In terms of the quality of minimally processed fruit, flesh browning is fundamentally important in the development of an aesthetically unpleasant appearance, with consequent off-flavours. The development of browning depends on the enzymatic action of the polyphenol oxidase (PPO). In the ‘Golden Delicious’ apple genome ten PPO genes were initially identified and located on three main chromosomes (2, 5 and 10). Of these genes, one element in particular, here called Md-PPO, located on chromosome 10, was further investigated and genetically mapped in two apple progenies (‘Fuji x Pink Lady’ and ‘Golden Delicious x Braeburn’). Both linkage maps, made up of 481 and 608 markers respectively, were then employed to find QTL regions associated with fruit flesh browning, allowing the detection of 25 QTLs related to several browning parameters. These were distributed over six linkage groups with LOD values spanning from 3.08 to 4.99 and showed a rate of phenotypic variance from 26.1 to 38.6%. Anchoring of these intervals to the apple genome led to the identification of several genes involved in polyphenol synthesis and cell wall metabolism. Finally, the expression profile of two specific candidate genes, up and downstream of the polyphenolic pathway, namely phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO), provided insight into flesh browning physiology. Md-PPO was further analyzed and two haplotypes were characterised and associated with fruit flesh browning in apple.     

Susceptibility of selected apple cultivars to the Mediterranean fruit fly

Ceratitis capitata (Wiedemann), the Mediterranean fruit fly, is one of the key pest species affecting deciduous fruit orchards along the Mediterranean coasts. Because of global warming, C. capitata is gradually spreading north and is becoming a major pest of apples. Determining the susceptibility of the main apple varieties grown in the region will serve as a cornerstone to the management of this pest. In this study, we show the results of a field and laboratory no‐choice test conducted to determine the Medfly preferences on different apple cultivars. The seven main varieties of apples (Gala, Red Delicious, Golden Delicious, Granny Smith, Kanzi, Morgen Dallago and Fuji) were tested. The results demonstrate that C. capitata lays eggs on all apple cultivars in both field and laboratory conditions. The Granny Smith, Red Delicious and Morgen Dallago varieties showed the lowest susceptibility in laboratory conditions, (0.75, 1.55, 2 oviposition punctures/fruit, respectively), with significant differences in oviposition compared to the Golden Delicious, Kanzi and Fuji (3.27, 3.31, 3.1 oviposition punctures/fruit, respectively) varieties, which were shown to be the most susceptible to Medfly attack in laboratory conditions. On the other hand, only slight and not statistically significant differences emerged from the field trials. In relation to the physico‐chemical characteristics, the apple cultivars showing the lowest susceptibility (Granny Smith, Red Delicious and Morgen Dallago) had harder peels and pulps and lower sugar contents than the most susceptible cultivars (Golden Delicious, Fuji and Kanzi). These results were also confirmed through evaluation of larval development on different varieties. In fact, Granny Smith, Red Delicious and Morgen Dallago were the three varieties that did not allow adequate larval and adult development and reduced the possibility of the emergence of a new generation.     

Blühfördernde Wirkung von 2-Chloräthylphosphonsäure bei Apfelbäumen

Experiments with the growth regulator ethephon (2-chloroethylphosphonic acid) were made to stimulate the flower differentiation in the ‘Carola’ and ‘Golden Delicious’ apple cultivars in Pillnitz (GDR) and ‘Jonathan’ and ‘Golden Delicious’ in Ujfehértó (Hungaria). A twofold application of 150 ppm ethephon in June proved most effective. Our results indicate that it is possible to promote flower differentiation in apple trees without growth retardation and fruit thinning.     

Laboratory tests carried out onMicrothrix inconspicuella [Lep.: Pyralidae], a biological control agent forEmex australis in Australia

Host specificity tests of the moth,Microthrix inconspicuella Ragonot in Australia, indicated that larvae could feed and develop on young apple leaves. Additional tests in South Africa on leaves and fruit of the 4 apple varieties, Jonathan, Starking (Red Delicious), Granny Smith and Golden Delicious, showed that apples were not a preferred food. Little feeding occurred and pupation happened infrequently. No 2^nd generation resulted whenM. inconspicuella colonies were confined on apple fruit or leaves.      

Growth of Escherichia coli O157:H7 in bruised apple (Malus domestica) tissue as influenced by cultivar, date of harvest, and source

Four of five apple cultivars (Golden Delicious, Red Delicious, McIntosh, Macoun, and Melrose) inoculated with Escherichia coli O157:H7 promoted growth of the bacterium in bruised tissue independent of the date of harvest (i.e., degree of apple ripening) or the source of the apple (i.e., tree-picked or dropped fruit). Apple harvest for this study began 4 September 1998 and ended 9 October, with weekly sampling. Throughout this study, freshly picked (<2 days after harvest) McIntosh apples usually prevented the growth of E. coli O157:H7 for 2 days. Growth of E. coli O157:H7 did occur following 6 days of incubation in bruised McIntosh apple tissue. However, the maximum total cell number was approximately 80-fold less than the maximum total cell number recovered from Red Delicious apples. When fruit was stored for 1 month at 4 degrees C prior to inoculation with E. coli O157:H7, all five cultivars supported growth of the bacterium. For each apple cultivar, the pH of bruised tissue was significantly higher and degrees Brix was significantly lower than the pH and degrees Brix of undamaged tissue regardless of the source. In freshly picked apples, changes in the pH did not occur over the harvest season. Bruised Golden Delicious, McIntosh, and Melrose apple tissue pHs were not significantly different (tree-picked or dropped), and the degrees Brix values of McIntosh, Macoun, and Melrose apple tissue were not significantly different. Single-cultivar preparations of cider did not support growth of E. coli, and the cell concentration of inoculated cider declined over an 11-day test period. The rate of decline in E. coli cell concentration in the McIntosh cider was greater than those in the other ciders tested. The findings of this study suggested that the presence of some factor besides, or in addition to, pH inhibited E. coli growth in McIntosh apples.     

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.     

Functional allelic diversity of the apple alcohol acyl-transferase gene <Emphasis Type="Italic">MdAAT1</Emphasis> associated with fruit ester volatile contents in apple cultivars

Flavour is an important key factor of apple (Malus × domestica Borkh.) fruit quality, and its improvement is an important but complex breeding goal. Acetate esters are quantitatively the most important volatile compounds in apple fruit, and only a few of them dominate the typical aroma of a cultivar. Alcohol acyl-transferase (AAT) is a key enzyme involved in the last step of ester biosynthesis. The aim of this study was to target single nucleotide polymorphisms (SNPs) in an AAT candidate gene genetically associated with ester quantitative trait loci (QTL), to enable functional marker development for marker-assisted apple breeding programs. The AAT gene inventory of apple was characterized by in-silico mining of the assembled Golden Delicious genome, and 17 putative AAT genes in total were defined. MdAAT1 located on chromosome 2 was selected as the main candidate gene associated with QTL for different acetate esters, and its allelic diversity was assessed by direct amplicon sequencing in a collection of 102 apple cultivars characterized for ester volatile profiles. Sequencing a 468 bp nucleotide sequence of the MdAAT1 coding region resulted in the detection of four SNPs. In total, 18 different SNP haplotypes/heterozygous patterns were generated from the four SNPs identified within the apple collection. Association analyses resulted in highly significant associations of both individual SNPs and distinct haplotypes with the content of four acetate esters, including hexyl acetate, butyl acetate and 2-methyl-butyl acetate. About a third (31) of the 102 apple cultivars possessed the specific MdAAT1 haplotype H1 (C-A-C-A) and were characterized by strongly decreased ester concentrations. The contrasting H8 haplotype (T-G-T-G) was found in 28 varieties but was associated with normal to elevated ester concentrations. The observed association suggests a putative causal functional relationship between MdAAT1 and production of key apple esters.