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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code HH3725STF

2. Project title

To optimise texture and flavour in stored apples using a genomic approach

3. Contractororganisation(s)

East Malling ResearchNew RoadEast Malling, KentKentME19 6BJ     

54. Total Defra project costs £ £536,576.00(agreed fixed price)

5. Project: start date................ 01 April 2004

end date................. 31 March 2008

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

Consumers and retailers require that all horticultural produce meet the highest standards for quality, reliability and availability. However, predicting and optimising the quality of stored fruit and maximising shelf life pose major problems to the industry, both in the UK and overseas. Currently, to extend the marketing of apple and other climacteric fruits, ripening processes are retarded by the use of refrigeration and controlled atmosphere (CA) storage. Approximately 80% of commercial UK apples are placed in CA each year. In apples, the rate of softening during storage can be partly controlled by these stringent CA conditions but the production of the fruit flavour compounds is reduced. Thus, it would be highly advantageous to uncouple the process of softening from flavour production in storage so that these could be controlled independently. Moreover, the energy and economic inputs into refrigerated CA storage are not consistent with Defra policy for the sustainable use of natural resources. Therefore, the main objective of this project was to use the unique genetic resources at EMR to develop the potential Despite much research, the role of specific genes and enzymes in the softening process is not yet known. An expression sequence tag (EST) library developed in a previous Defra project (HH1022) was used to help identify softening-related genes. Primers were designed to clone fragments of several softening-related genes including β-galactosidase, endo-polygalacturonase, endo-xyloglucan hydrolase 5 (XET5), pectin methyl esterase and pectate lyase from genomic DNA extracted from the Malus pumila cultivars ‘Fiesta’ and ‘Totem’. All fragments were sequenced and have been added to the ‘Fiesta’ × ‘Totem’ linkage map. At least two of these candidate genes appear to co-locate with putative QTLs for firmness.

Once softening-related ESTs had been mapped, the expression of these genes during the initiation of softening in ‘Cox’ apples and in apple lines with different softening characteristics was determined using Real Time–PCR. The expression of the softening-related genes XET5, β-galactosidase (4), β-galactosidase (8), pectate lyase and endo-polygalacturonase was increased after exposure to ethylene during the initiation of softening in ‘Cox’ apples. The accumulation of β-galactosidase (8) and endo-polygalacturonase transcripts was much lower in the non-softening lines ‘COOP 44’ and ‘D113’. These genes encode cell wall degrading enzymes and the delay and overall reduction in their transcription contributed to the maintenance of firmness in ‘COOP 44’ and ‘D113’ apples under conditions conducive to softening.

The potential of apple lines with a heritable trait for low ethylene production to be stored under low stringency conditions (in air at higher than recommended temperatures) was determined. If successful, this approach could be developed as a low input, sustainable alternative to CA storage to maintain fruit texture and flavour. Low ethylene producing lines stored well in air at higher storage temperatures (5 °C) than in current CA regimes without unacceptable losses in firmness. Application of 1-MCP (an inhibitor of ethylene action) further increased the storage life of low ethylene producing apples. However, in apple lines with very

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low ethylene production rates, flavour volatile production and fruit acidity were reduced and the incidence of rotting during storage was higher. Therefore, a threshold level of ethylene production is needed to maintain quality during storage and it will be important to distinguish between ‘low’ and ‘very low’ ethylene producing lines in any future efforts to improve the storage potential of apples.

Previous work (Defra HH2606STF) showed that the low ethylene trait could be partly explained by a mutated, ripening-specific ACC synthase (ACS) gene (MdACS-1). However, another, as yet unknown gene must underlie the different ethylene production rates of the low ethylene lines. The D-family apple progeny have already been categorised on the length of time taken to produce ethylene (1 nL g-1 h-1) during storage (HH2606), and bulk segregant analysis was used on this progeny to try to identify additional markers linked to other component(s) of the low ethylene trait. In addition to a major QTL for low ethylene spanning MdACS-1, MdETR-1 and three SSRs on linkage group 15 (LG15), a ‘non ACS-1’ QTL explaining 15% of the variance was also detected on LG15. The three SSRs (CH02d11, CH03b06 and CH03h03), heterozygous in both D-family parents, mapped to the region explaining 15% of the variance for the trait and could be used to aid future marker-assisted selections.

Apples with improved storage characteristics will eventually facilitate the use of less stringent, more sustainable storage regimes that optimise fruit quality. The principal progenies already available for the development of markers co-segregating with storage traits were those segregating for low ethylene production which were mapped in Defra HH2606STF and those from the 'Fiesta' × 'Totem' cross (Defra HH1029STF). To increase the portfolio of useful markers for further traits, additional progenies were raised for future mapping work. A range of crosses was made among nine different varieties with differing storage capacities. Three progenies were raised that will produce fruit of varying keeping qualities for assessment in a future project and thus enable the mapping and marker development for these key traits. The progenies ‘E811’, ‘E830’ and ‘E832’ will facilitate future work aimed at improving the sustainability of storage regimes for UK produced top fruit. Such material will eventually benefit both growers and consumers, by enabling the use of cold storage, rather than CA storage, to provide fruit at point of sale that matches consumer expectations. EMR is currently evaluating how best to exploit these unique genetic resources.

The results of the project have been transferred to the grower community through EMRA Storage Days, the trade press and to the academic community at international conferences and through peer-reviewed publications in scientific journals. However, due to changes in Defra Policy Areas, a shift in emphasis in EMR’s science strategy towards soft fruit and only moderate industry support, it was not feasible to assemble a consortium to develop a project proposal to Horticulture LINK. Discussions will be held with key representatives from the top fruit sector during 2008 to determine whether improving storage potential and reducing the costs and inputs associated with high stringency CA regimes are likely to be an industry priority in the short-term.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

IntroductionThe value of fruit crops is maximised when they are at the optimum stage of ripeness at the point of sale. Although approximately 80% of UK apples go through controlled atmosphere (CA) storage prior

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to sale, it remains difficult to achieve consistently high quality at harvest, during storage, and through the supply chain. Understandably, the storage of apples and other climacteric fruit is a prime concern for UK producers and marketers. In apples, the rate of softening during storage is particularly sensitive to ethylene and can be partly controlled by refrigeration (0-4 oC) and controlled atmospheres (1-3% O2, 5-10% CO2). However these stringent CA conditions used to retard softening interfere with the production of the fruit flavour compounds that is another essential requirement for consumer acceptance. Thus, it would be highly advantageous to uncouple the process of softening from flavour production in storage so that the two could be controlled independently.

The main objective of this project was to use the unique genetic resources at EMR to develop the potential to improve texture and flavour attributes of stored apples while reducing the costs and energy inputs associated with CA storage.

Scientific Objectives1) To use an apple EST library to develop markers for softening-related genes and to map these to

the apple genetic linkage map.2) To determine expression of softening-related ESTs in apple lines with different softening

characteristics.3) To use several low and very low ethylene producing lines to determine the potential of using

refrigeration at higher than normal temperatures, with less stringent CA storage, to optimise texture, flavour production and alleviate storage disorders.

4) To identify microsatellite markers linked to 'non ACS-1 traits' determining ethylene production to aid marker-assisted selection in the industry-funded breeding programme.

5) To raise new progenies derived from cultivars contrasting for a range of good storage attributes for future mapping of these traits to permit marker-assisted selection in the industry-funded breeding programme.

6) To communicate the results of this study to the grower and academic communities and to develop a Horticulture LINK consortium to facilitate effective technology transfer to the horticultural industry.

Progress in relation to stated objectivesObjective 1 was fully met. Gene-specific markers were developed for the softening-related genes β-galactosidase, endo-polygalacturonase, endo-xyloglucan hydrolase 5 (XET5), glutathione peroxidase, pectin methyl esterase and pectate lyase; these genes have been added to the ‘Fiesta’ × ‘Totem’ linkage map. At least two of these candidate genes appear to co-locate with putative QTLs for firmness.

Objective 2 was fully met. Transcripts of XET5, β-galactosidase (4), β-galactosidase (8), pectate lyase and endo-polygalacturonase all increased after exposure to ethylene during the ‘initiation of softening’ period. The accumulation of β-galactosidase (8) and endo-polygalacturonase transcripts was much lower in the non-softening lines ‘COOP 44’ and ‘D113’. These genes encode cell wall degrading enzymes and the delay and overall reduction in their transcription contributed to the maintenance of firmness in ‘COOP 44’ and ‘D113’ apples under conditions conducive to softening.

Objective 3 was fully met. Low ethylene producing lines have the potential to be stored in air at higher storage temperatures (5 °C) than in current CA regimes without unacceptable losses in firmness. Application of 1-MCP (an inhibitor of ethylene action) further increased the storage life of ‘low ethylene’ producing apples. However, air storage reduced concentrations of organic acids in these fruit and the fall in acidity could not be prevented by lowering the oxygen concentration in store to 2%. Flavour volatile production was impaired in the very low ethylene lines and the incidence of rotting was generally higher in these fruit. Therefore, a threshold level of ethylene is necessary to maintain quality during CA storage and it will be important to distinguish between ‘low’ and ‘very low’ ethylene producing lines in any future efforts to improve the storage potential of apples.

Objective 4 was fully met. Microsatellite markers and candidate genes linked to the 'non ACS-1’ trait determining late ethylene production were identified and will aid future marker-assisted selections.

Objective 5 was fully met. Novel breeding lines with different storage potentials have been raised and will facilitate future research into quality attributes of stored fruit. A thorough analysis of fruit

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organoleptic quality in these progenies will form an important component of any follow-on project to improve the sustainability of UK top fruit storage.

Objective 6 was partially met. The results of the project have been transferred to the grower community through EMRA Storage Days, the trade press and to the academic community through a peer-reviewed publication in an international scientific journal. However, due to changes in Defra Policy Areas, a shift in emphasis in EMR’s scientific strategy towards soft fruit and only a moderate opportunity for industry financial support, it was not feasible to assemble a consortium to develop a project proposal to Horticulture LINK.

Objective 1IntroductionSoftening of fleshy fruit is a quantitative trait involving numerous loci (Fulton et al., 2000; King et al., 2000), and therefore, many gene products. Despite much research, the role of specific genes and enzymes in this process is not yet known. Recently, two MADS-like FUL orthologues associated with softening in apple have been identified and mapped as part of HH3701SX. Rather than focusing on specific genes such as FUL, a genomic approach was used in this project to study the softening process in apple. A collection of expressed sequence tags (ESTs) from cDNA libraries prepared from RNA extracted from pre- and post-climacteric apples was used. Approximately 440 of these ESTs have already been sequenced (HH1022) and some of these genes have a known role in cell wall disassembly. The remaining ESTs were sequenced to identify additional genes associated with cell wall biosynthesis and disassembly. These candidate genes were integrated into a functional map of the apple genome and were tested for their association with quantitative trait loci (QTLs) identified both within this project and from those putatively identified in the EU-funded HiDRAS project (High quality Disease Resistant Apple for a Sustainable agriculture).

Materials and MethodsPrimers were designed to clone fragments of cell wall synthesis and softening-related genes from genomic DNA extracted from Malus pumila cultivars ‘Fiesta’ and ‘Totem’. These included: β-galactosidase, endo-polygalacturonase, XET5, pectin methyl esterase (PME), pectate lyase, cellulose synthase, dolichyl-di-phospho-oligosaccharide and proyl-endopeptidase. Other ripening-related genes involved in flavour, sweetness and acidity (alcohol dehydrogenase, aldehyde dehydrogenase, sorbitol dehydrogenase, lipoxygenase, alpha-mannosidase, glutathione peroxidase and malate dehydrogenase), cell signalling (MAP kinase ‘yabby 2 like’ ethylene induced transcription factor, a calcium ion channel protein [Ca2+ ATPase]), fruit quality (alpha-farnesene, Maldi 1) and two genes that have a role in combating stress (metallothionein protein and dehydrin/ABA dehydration response protein) were also cloned from ‘Fiesta’ and ‘Totem’ genomic DNA. All fragments were sequenced and alignments between ‘Fiesta’ and ‘Totem’ clones yielded some regions of polymorphism.

Moreover, labelled ‘pig-tailed’ primers from softening/ripening genes were designed around regions of polymorphism and microsatellite markers were identified by aligning Malus EST sequences against homologous Arabidopsis genomic and cDNA gene sequences (http://www.tair.org/), thus allowing primers to be designed against intron/exon (supposedly functional and non-functional regions of DNA) boundaries.

The amplification data from these primers on the ‘Fiesta’ × ‘Totem’ progeny were collected using an ABI 3100 Genetic Analyzer with an internal size standard (ROX™ or 500 LIZ™) using GENESCANâ

3.7 and GENOTYPERâ 3.7 software (Applied Biosystems). The data were then mapped onto the ‘Fiesta’ × ‘Totem’ reference map (Fernández-Fernández et al., 2008) using the software JOINMAP® 4.0 (Van Ooijen, 2006).

ResultsA total of 960 softening/ripening related EST’s were analysed using ncbi/tigr/tair blast software packages. Endo-polygalacturonase, dolichyl-di-phospho-oligosaccharide, malate dehydrogenase, Ca2+

ATPase, aldehyde dehydrogenase and alcohol dehydrogenase were mapped onto the ‘Fiesta’ × ‘Totem’ linkage map. Other genes including pectate lyase, XET5, glutathione peroxidase and auxin binding protein were also placed on the ‘Fiesta’ × ‘Totem’ linkage map.

Pectate lyase maps towards the bottom of linkage group 14 (LG14) which is also the putative position of a ‘firmness at harvest’ QTL identified in HiDRAS. Another co-location is that of glutathione peroxidase, which maps towards the top of LG6, within a ‘firmness two-months post-harvest’ HiDRAS putative QTL.

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DiscussionAn EST library developed in a previous Defra project (HH1022) was used to help identify softening-related genes. Primers were designed to clone fragments of softening-related genes β-galactosidase, endo-polygalacturonase, XET5, glutathione peroxidase, PME and pectate lyase from genomic DNA extracted from the Malus pumila cultivars ‘Fiesta’ and ‘Totem’. All fragments were sequenced and have been added to the ‘Fiesta’ × ‘Totem’ linkage map. At least two of these candidate genes appear to co-locate with putative QTLs for firmness. Interestingly, the two MADS-like FUL orthologues identified and mapped as part of HH3701SX by the group at Warwick-HRI also appear to map to the same linkage groups in approximately the same positions.

Objective 2IntroductionTo help determine whether the candidate genes identified under Objective 1 are involved in the softening process, the expression of these genes was determined in pre- and post-climacteric ‘Cox’ fruit and ‘COOP 44’ and ‘D113’ apples, two lines with different softening characteristics using Real-Time PCR. These expression profiles were then compared to changes in firmness measured with a penetrometer and also wedge fracture tests throughout the softening process in ‘Cox’ and in ‘COOP 44’ and ‘D113’ lines that maintained firmness under conditions conducive to softening.

Materials and Methods‘Cox’, ‘Bramley’ and ‘Gloster 69’ fruit were harvested on 10 September 2004 and stored at 20 ºC in a flow of air (1 L kg-1 h-1) amended with 200 ppm of ethylene. At intervals, fruits were removed and firmness measurements made using a penetrometer fitted with an 11 mm probe. In 2005, the experiment was repeated with ‘Cox’ apples alone, while in 2006 ‘Cox’ and two non-softening apple lines ‘D113’ and ‘COOP 44’ were harvested on 4 September and stored at 20 ’ºC while treated with ethylene as described above. In 2007, penetrometer readings were followed by removal of 1 cm3 cube of tissue excised from opposing equatorial regions of apple and then subject to wedge fracture tests. After measurements of firmness, samples of cortex were flash frozen in liquid nitrogen and stored at -20°C. RNA was extracted from apple cortex under standard conditions. The concentration and purity of RNA samples was measured using A260 and A280 , aliquots of RNA were treated with DNase I for 10 min at room temperature, before being deactivated at 60°C for 10 min. The integrity of RNA was checked by separating samples on a denaturing RNA gel. cDNA was synthesised from 3 µg samples of RNA using an ABI archive kit using random hexamer primers. A subtractive enriched cDNA library (EST) prepared from apple cortex that had been exposed to 200 ppm of ethylene and representing gene transcripts from the initial stages of cortex softening had been designed in a previous Defra project (HH1022). Further ESTs from this library were sequenced leading to the analysis of 960 EST using ncbi/tigr/tair blast software packages.

Initially, a large scale screening of ESTs to identify genes up-regulated or down-regulated during the early stages of apple softening was attempted using a nylon-filter ‘macro-array’ technique. Six hundred plasmids (TOPO ®? pCr 2.1) containing ESTs were amplified by PCR using universal M13 primers flanking the insert. The PCR products were dot-blotted onto nylon-filters in triplicate and fixed to the membrane by exposure to UV light. DNase treated RNA extracted from ‘Cox’ apple cortex previously exposed to 200 ppm of ethylene for zero, two or five days was used as a template to synthesise Dig-labelled cDNA following standard protocols (Roche). Dig-labelled cDNA was used to probe membranes containing ESTs, followed by chemiluminescence detection (Roche). Although the technique was sufficiently sensitive to detect up-regulation of genes such as ACC oxidase and endo-polygalacturonase, the method wasn’t sufficiently sensitive to identify many of the EST library. Therefore, Real-Time PCR was used to screen approximately 100 ESTs representing genes that may have a role in cell wall softening, cell turgor or genes that were likely to be involved in cell signalling or hormone responsive genes. Primers were designed to amplify a 100-150 bp amplicon to maximise amplification efficiency during Real-Time PCR, two endogenously expressed genes, beta-actin and ITS region of 18S ribosomal RNA were used as internal controls. Three replicate samples (25 ng) of cDNA were used to investigate up and down regulation of genes selected from a subtractive EST library using Real-Time PCR (ABI) using a standard program (1 cycle of 95 °C for 10 min, 40 cycles of 94 °C for 15 s, 60 °C for 1 min); fluorescence was measured at the 60 °C stage in the PCR cycle.

ResultsAnalysis of subtractive EST library

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5% 2%17%

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Figure 1. Sequence homology of subtractive enriched EST library prepared from ‘Cox’ apples exposed to 200 ppm of ethylene for two days and stored at 20 °C.

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Figure 2. Relative expression (2-ΔΔCT) of softening-related genes in ‘Cox' apples stored in air at 20 °C and exposed to 200 ppm of ethylene for 0, 2 and 5 days.

The proportion of sequences with homology to Malus and other rosaceous species was determined (Figure 1). A large proportion (55%) of the sequence data had no or low homology to known sequence identities. A total of 5% of the sequences were homologous to Malus species with a further 2% homologous to other members of the rosaceous family, 17% of sequences were homologous to Arabidopsis and 19% to other plant species. Sequences with homology to enzymes with reported involvement in cell wall disassembly or cell-to-cell cohesion were identified. β-galactosidase and endo-polygalacturonase were two enzymes most represented in the library. XET5, PME and pectate lyase were also identified as transcripts that have a role in softening.

Desiccation response transcripts (e.g. dehydrin, aldehyde dehydrogenase) were also highly represented in the library. A large number of transcripts was identified as having a role in cellular communication, signal transduction and initiation of transcription (transcription factors) (Annex 1, Table 1).

Expression profile of softening/ripening related ESTs in ‘Cox’ applesIn the first two years of the project, time-course experiments were conducted to determine the length of time taken to ‘initiate softening’ in ‘Cox’ apples during storage at 20 °C in the presence of 200 ppm of ethylene. The period before any physical sign of softening was detected, termed the ‘initiation of softening’, varied between years but ranged between two and five days after exposure to conditions conducive to ripening.

Real-Time PCR analysis of ‘Cox’ apples sampled from 2004 using methods described by Yuan et al. (2006), showed an increase in relative gene expression (2-ΔΔCT) in a number of cell wall enzymes responsible to cell wall modifications (Figure 2). XET5, responsible for transfer of xyloglucan components to other xyloglucans as well as expressing hydrolytic activity, showed a six-fold increase in expression. β-galactosidase (4), which cleaves β-(1→4)-galactosidic linkages in cell wall glycans, showed a doubling in expression, while its isoform β-galactosidase (8) showed a 120-fold increase. Transcripts of pectate lyase, which encodes enzymes that cleave α-(1→4) links of the polygalacturonan chain by β-elimination, showed a 14-fold increase in expression over five days exposure to ethylene. Endo-polygalacturonase, which depolymerises the pectic backbone of the cell wall matrix, increased over 60-fold after exposure to ethylene. The expression of a putative auxin binding protein/receptor also increased. Changes in relative gene expression of other genes involved in turgor-related processes, synthesis of flavour compounds and components of cell signalling were also detected (Annex 1, Table 1).

The experiment was repeated in 2005/06, in this year although fruits were harvested in the normal September harvest window for ‘Cox’, fruits were noticeably softer than in the previous year suggesting that some softening had already occurred prior to harvest. Thus, the difference in the degree of softening before and after exposure to ethylene was less pronounced than in other years. Presumably, endogenous ethylene had already induced transcription of some genes prior to addition of exogenous ethylene and so subsequent increases in transcription were less obvious. However, increase expression of endo-polygalacturonase, β-galactosidase isoforms 4 & 8, pectate lyase, elongation factor (EG3) and auxin binding protein were observed over the 11 day period (Annex 1,

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Figure 3. Relative gene expression (2-ΔΔCt) of selected EST’s with a role in cell wall disassembly in A) ‘Cox’s Orange Pippin’, B) ‘COOP 44’ and C) ‘D113’ apples. Fruit were stored under a flow of air amended with 200 ppm of ethylene at 20°C for up to 19 days in September 2007. Note different x-axis scales in A) and B) and C).

Table 2). Dehydrin and aldehyde dehydrogenase gene transcripts also increased in response to ethylene; both are known to increase in response to ethylene and moisture loss. As expected, alpha-farnesene, ACC oxidase and ACC synthase acted as positive controls; each are known to increase in response to ethylene and all showed an increase in transcript abundance over the 11 days exposure to ethylene.

In 2006/07, ‘Cox’ and two non-softening apples ‘COOP 44’ and ‘D113’ were harvested on 4 September and placed immediately into 20 °C with the addition of 200 ppm of ethylene. Apples were removed at intervals and measurements of firmness were made with a penetrometer and wedge fracture tests (Annex 1, Table 3). A comparison of the two techniques demonstrated that wedge fracture tests were more sensitive to changes in texture than standard penetrometer measurements. ‘Cox’ apples started to soften after five days of treatment with firmness dropping from 77.8 N at day five to 40.6 N after 19 days. In comparison, Work of Fracture (WOF) measurements dropped from 424.4 N/m to 74.3 N/m. In contrast, ‘COOP 44’ apples only softened by 8 N over the same period and WOF measurements fell by 73.2 N/m. ‘D113’ apples only softened from 133.0 N to 128.8 N over the first nine days but after another ten days the firmness dropped to 76.6 N. WOF measurements dropped from 592 N/m at day zero to 178 N/m by day 19.

These changes in firmness of ‘Cox’, ‘COOP 44’ and ‘D113’ apples were then compared to a range of genes that showed changes in expression profiles in previous years. In ‘Cox’, the biggest change was in the expression of endo-polygalacturonase where expression increased over 4,000-fold after five days then dropped to just under 2000-fold at day nine (Figure 3A). This was a significantly large increase in expression, not previously seen in other experiments. The abundance of β-galactosidase (8) transcript increased 60-fold by day five and up to 80-fold by day nine. XET5 also showed a small burst in activity after two days but then no further increase was observed.

In comparison, endo-polygalacturonase activity in the non-softening ‘COOP 44’ and ‘D113’ increased after exposure to ethylene but the rate of increase was much lower and the peak in activity appeared after 19 days exposure to ethylene (Figure 3 B&C). β-galactosidase transcript activity in ‘COOP 44’ apples also increased to a similar extent to that in ‘Cox’ apples but again the peak in transcription was ten days later. ‘D113’ apples also exhibited an increase in β-galactosidase activity but it was significantly less than in ‘Cox’ and ‘COOP 44’ apples.

In ‘Cox’, auxin binding protein transcripts increased significantly after five days and continued to increase up to day 19 where they were 121-fold higher than day zero (Figure 3). Auxin binding protein transcript activity in ‘COOP 44’ apples increased with exposure to ethylene and exhibited a similar pattern of expression to ‘Cox’ apple but the increase in ‘D113’ apples was significantly higher (Figure 3). Dehydrin also increased in ‘Cox’ with a burst of expression at day five followed by a decline in expression. Dehydrin transcript activity increased in ‘COOP 44’

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apples after two days exposure to ethylene with a second larger burst of activity by day 19. In ‘D113’, dehydrin expression increased by day nine but the abundance of transcripts was significantly less than ‘Cox’ or ‘COOP 44’ apples (Figure 3). The abundance of transcripts for pectate lyase, XET5 and PME remained unchanged in all three apple lines sampled in 2006 over the 19 days exposure to 200 ppm of ethylene (Figure 3).

DiscussionExpression patterns of the softening-related genes mapped under objective 1 were determined in ‘Cox’ apples at different stages during ripening under conditions conducive to softening. Transcripts of XET5, β-galactosidase isoforms 4 and 8, pectate lyase and endo-polygalacturonase all increased after exposure to ethylene during the ‘initiation of softening’ period. Interestingly, expression of a putative auxin binding protein/receptor also increased in ‘Cox’ apple tissue undergoing softening. Auxins are known to increase cell wall elasticity and are involved in pollination, fruit set and ripening; auxin signalling appears to be a prerequisite for cell enlargement in tomatoes (Seymour et al., 2008). The increase of an auxin binding protein after exposure to ethylene suggests that cross-talk between hormones plays an important role in fruit ripening.

Numerous proteins have been identified in cell walls that provide structural support such as extensins, while hydrolases and expansins degrade the cell wall. Glycoproteins make up the biggest component of cell wall proteins and include hydroxyproline-rich glycoproteins (HRGP’s) and arabinogalactans (AGP’s); these two proteins have a role in cell differentiation and cell development. Proteases including endo-prolylpeptidase, a cytosolic proline specific serine protease which can attack proline-rich proteins such as HRGP’s, were identified in the subtractive EST library and found to be highly up-regulated in ‘Cox’ tissue exposed to ethylene. The rate at which new cell wall material is laid down may alter the rate of softening, so genes involved in the synthesis of new glycans were also investigated. However, ethylene did not alter the rate of transcription of genes encoding enzymes involved in the synthesis of new cell wall material such as dolichyl diphospholigosaccharide, cellulose synthase and UDP glucose flavanoid 3-0 glucosyl transferase.

Apart from texture, flavour is also an important attribute to fruit quality. Sorbitol is used by Malus species to transport sugars from leaves to fruit where it is broken down into fructose and glucose by sorbitol dehydrogenase. The six-fold increase in sorbitol dehydrogenase transcription detected after exposure to ethylene could be expected to increase the amount of available monosaccharide sugars in the fruit. Lipoxygenases break down lipids and fatty acids and some of these breakdown products feed into flavour volatile synthesis. Lipoxygenase transcripts increased ten-fold after five days exposure to ethylene. Aldehyde dehydrogenase is involved in the formation of esters, which are important flavour components and it is also induced under conditions of moisture loss and may help to maintain cell turgor under unfavourable conditions. Transcripts encoding alpha-farnesene were also highly up-regulated after exposure to ethylene; alpha-farnesene formation is an important intermediate in the formation of superficial scald in that it is oxidised by free radicals to form conjugated trienes. These trienes oxidise membrane lipids leading to localised cell death and a browning of epidermal cells. Although alpha-farnesene is actively synthesised in ‘Cox’ apple tissue, superficial scald is rarely observed as a physiological disorder in this variety, thus confirming that oxidation to conjugated trienes is the rate-limiting step in the formation of scald in ‘Cox’.

Gene transcripts of cell signalling components were also investigated throughout the period of softening in ‘Cox’ apples. The MADS box family of transcription factors are important in regulating fruit development and ripening in Angiosperms. Fruitfull (FUL) is a member SQUAMOSA class of MADS box transcription factors that is involved in carpel development and has an overlapping function in controlling ripening (Seymour et al. 2008). FUL is a negative regulator of ripening so a decrease in its expression may signal a cascade of ripening events. Although FUL was not identified in the subtractive library, its role in controlling ripening required its inclusion in this study. Expression of FUL declined with increasing fruit maturity, while the expression of other transcription factors remained constant. The abundance of MAP kinase and ACC oxidase-I transcripts increased after exposure to ethylene indicating that positive feed-back mechanisms were actively synthesising more ethylene.

The expression of softening-related genes in apple lines with different softening characteristics was also determined. The maintenance of firmness in ‘COOP 44’ and ‘D113’ apples under conditions conducive to softening may, in part, be due to delayed transcription of genes encoding cell wall degrading enzymes combined with a reduced amount of overall transcription. β-galactosidase (8) and endo-polygalacturonase play a significant role in cell wall disassembly and were transcribed in significant quantities in ‘Cox’ apples during softening. However, the accumulation of these transcripts was much lower ‘COOP 44’ and ‘D113’ over the same period. Shakespeare (2001) also concluded

SID 5 (Rev. 3/06) Page 10 of 20

that elevated activity of β-galactosidase increased the rate of softening in ‘Cox’ apples during storage. The lack of pectate lyase, XET5 and PME activity may have been due to the maturity of the fruit at harvest. Transcripts for XET5 undergo a transient rise in young expanding fruit and again during the climacteric (De Silva, 1994), and it is possible that increases in transcription of some enzymes involved in softening may have occurred earlier during fruit development.

Objective 3IntroductionFlavour development in apple takes place over a period of days or weeks both during ripening on the tree and throughout subsequent storage. Currently, apples are picked in a pre-climacteric state and stored under CA conditions to inhibit softening. Although commercial storage regimes used to inhibit softening of stored apples preserve the acid content of fruit, they greatly reduce the production of flavour volatiles. If low ‘ethylene’ producing lines could be used to ensure that firmness was maintained, it may be possible to delay harvesting to allow apples to develop fuller flavour on the tree before storage in low stringency CA regimes at higher temperatures than currently possible. Esters are the major class of compounds found in the flavour volatiles of apples, and in many other dessert fruits. In apple, these include hexyl acetate, amyl acetate and butyl acetate. The largest change in volatile emission during ripening is due to an increase in ester production (Brown et al., 1966) which is inhibited when ethylene action is blocked (Fan & Mattheis, 1999). In melons that had been genetically transformed to reduce ethylene synthesis, ester formation was depleted (Bauchot et al., 1998). Thus, the production of flavour volatiles seems to be linked to ethylene perception and/or ethylene action. Therefore, we tested whether flavour volatile production was limited in the low ethylene lines.

Currently the UK apple industry relies on sophisticated and costly methods of storage to retard ethylene-dependent ripening processes such as softening and the development of physiological disorders such as superficial scald and senescent breakdown. New cultivars that possess traits for low-ethylene production will provide major benefits to consumers, retailers and growers without the need for post-harvest chemicals. However, it is important to identify any potential adverse effects associated with an altered ethylene metabolism. For example, low ethylene storage of ‘Cox’ and ‘Bramley’ apples can increase the incidence of physiological disorders (Johnson & Colgan, 2003). It is likely that a threshold level of ethylene production is necessary to prevent the development of storage disorders. Therefore, we tested whether the low ethylene trait predisposed fruit to physiological disorders during storage.

Materials and methodsLow ethylene producing apples from a ‘Fiesta’ × ‘Gloster’ cross (‘E19/13’) and an un-named selection × ‘Gloster 69’ (‘E55/55’) were harvested on the 26 September 2004, 27 September 2005 and 28 September 2006. Fruits were randomised and ten apples per replicate were placed into nets and air-stored at 3.0-3.5 °C in 2004/05, 1.5-2.0 °C in 2005/06 and at 5.0-5.5 °C in 2006/07. At monthly intervals, two replicate samples of ‘E19/13’ and ‘E55/55’ were removed from store and firmness measurements were taken on whole apple samples using a penetrometer. Wedge-test measurements were then conducted on 1 cm3 cubes of cortex tissue excised from opposite sides of each apple.

The use of the ethylene action inhibitor 1-methylcyclopropene (1-MCP) to increase the storage life of low ethylene producing fruit was also investigated. Trials were conducted with low ethylene producing lines ‘E19/13’, ‘E55/55’ and ‘Gloster 69’. Apples were harvested on 19 September 2006, randomised and ten apples per replicate were placed into nets. Half the netted samples were treated with 625 ppb of 1-MCP at 20 °C for 24 h while the untreated fruit were kept at 20 °C for 24 h. Treated and untreated fruits were further divided into two subsets; one set were stored in air at 3.5 °C while the other were stored in air at 5 °C. At monthly intervals between September 2006 and March 2007, two replicate samples of ‘E19/13’, ‘E55/55’ and ‘Gloster 69’ were removed from each storage regime and firmness measurements were taken.

In addition to the trials with air-stored low ethylene producing fruits, the use of low-stringency CA storage was also investigated as a method of maintaining texture and flavour profiles during storage. Of particular interest was the effect of storage environment on fruit acidity. In 2005/06 and 2006/07, the low ethylene producing apples ‘E55/55’, ‘E19/13’ and ‘Gloster 69’ were stored in either 21% O2 (air), 5% O2,, 3% O2 and 2% O2 at 1.5 °C. Fruits were removed from storage in January, March and May. For organic acid analyses, 3 ml of juice were reduced with 30 μl of 500 mM tris (2-carboxyethyl) phosphine hydrochloride. Three hundred microlitres were then transferred to a plastic HPLC vial, and 300 μl of 20 mM K3PO4 (pH 2.9) buffer was added. Each vial was capped with a syringeless filter device to remove any debris from the samples which could block the HPLC column.

SID 5 (Rev. 3/06) Page 11 of 20

Figure B

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Figure 4. The decline in A) work of fracture, B) maximum load and C) firmness of low ethylene producing apples (‘E19/13’ and ‘E55/55’) during air-storage at 3-3.5 °C. Measurements of work of fracture and maximum load were conducted using a ‘wedge-test’ while firmness of whole apples were made with a penetrometer with a 11 mm probe.

Organic acids were quantified using a Waters Alliance 2690 HPLC equipped with a photodiode array detector (PDA). Sample extracts (10 μl) were run isocratically through a 4 μm 150 × 2.0 mm Synergi Hydro-RP 80A column fitted with a SecurityGuard cartridge. The mobile phase was 20 mM potassium phosphate buffer (pH 2.9), the column flow rate was 0.15 ml min-1, and the temperature was maintained at ambient.

For sugar analyses, 300 μl samples were transferred to plastic HPLC vials and capped with a syringeless filter device. Sugars were quantified using a Waters Alliance 2690 HPLC equipped with a refractive index detector (RID). Sample extracts (10 μl) were run through a Pinnacle II amino 5 μm 150 × 4.6 mm column. The mobile phase was 80% methanol, the column flow rate was 1 ml min -1, and the temperature was 35 °C. The production of important apple flavour volatiles in the low ethylene lines was also determined. Apple volatile components change rapidly postharvest and after freeze-thawing, so only fresh samples were used. Fruit samples were harvested at 09:30 and transported immediately to Reading Scientific Services Limited, where the flavour volatile content was analysed the same day using headspace gas chromatography-mass spectrometry (GC-MS). Samples were analysed under nitrogen using a purge-and-trap headspace technique, and then by automated thermal desorption (ATD) linked GC-MS. The compounds from the resulting flavour volatile profiles were identified and quantified against a general internal standard (ethyl benzene d10). Concentrations were divided by published ‘odour threshold of detection concentrations’ to give 'Odour units'. Volatile compounds with an odour unit equal or greater than 1 are thought to contribute to apple flavour (Guadagni et al., 1966).

D-family apple progeny have already been categorised on the length of time taken to produce ethylene (1 nL g-1 h-1) during storage (HH2606STF). Ten progeny from four sub-sets of apples classed as early producers (<15 days), mid-term producers (130-150 days), mid-late term producers (170-220 days) and late producers (>250 days) were selected. Four ten-fruit samples were taken from each category and placed into nets. Two replicates received 625 ppb of 1–MCP at 20 °C for 24 h while the remaining two replicates were left untreated. This trial was repeated over three years and apples were stored in air at a different temperature each year (1.5-2.0 °C, 3.0-3.5 °C and 4.5-5.0 °C) for four months. There were no significant differences in the effects of temperature on fruit firmness or the incidence of disorders so data from apples stored at 1.5-2.0 °C are presented.

ResultsCan the firmness of low ethylene producing apples be maintained in air storage alone?Experiments were conducted over three years to determine whether low ethylene producing apples could be maintained in air storage alone, without the need for costly CA storage. ‘E19/13’ and ‘E55/55’ apples were air-stored at different temperatures during 2004/05, 2005/06 and 2006/07 to determine whether these low ethylene producing lines could be stored under low stringency conditions without unacceptable losses in firmness. At each harvest, firmness of ‘E55/55’ measured using a standard penetrometer was higher than that of ‘E19/13’ but ‘E19/13’ fruit softened more slowly over the six-month storage period (Figure 4). Similar results were obtained in 2005/6 and 2006/07, despite different storage temperatures.

Wedge test measurements on the two apple lines exhibited a similar pattern of softening to that determined by penetrometer measurements (Figure 4C). However, work of fracture (WOF) measurements provided greater discrimination in firmness data compared to the penetrometer. Correlation between penetrometer and WOF measurements was greater with ‘E55/55’ apples (0.980) than with ‘E19/13’ (0.667) and similarly the correlation with maximum load data using a wedge test was better with ‘E55/55’ (0.977) than with ‘E19/13’ (0.719).

SID 5 (Rev. 3/06) Page 12 of 20

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Figure 6. The effect of different oxygen concentrations during storage for A) 4 months and B) 6 months on fruit firmness of low ethylene lines during storage at 1.5 °C, Measurements of firmness were made using a penetrometer with a 11 mm probe. LSD0.05 =5.3 on 24 df.

The use of 1-MCP to increase the storage life of low ethylene producing fruit was also investigated. 1-MCP was more effective than temperature alone in preventing softening in apples. In certain apple lines, there was an additive effect of temperature and 1-MCP. Unexpectedly, the firmness of ‘E19/13’ apples (Figure. 5A) stored at 3 °C declined (36 N) to a greater extent than those stored at 5 °C (14 N). In contrast, ‘E19/13’ apples treated with 1-MCP and stored at 3 °C only softened by 2 N, compared to 14 N in treated fruit stored at 5 °C. Temperature had no effect on the decline in softening in ‘E55/55’ apples (Figure 5B) although firmness tended to be higher at 5 ºC for most of the storage period. However, 1-MCP halved the rate of softening in apples stored at 3 °C or 5 °C, both sets of 1-MCP treated fruits softened at the same rate. The rate of softening in untreated ‘Gloster 69’ apples (Figure 5) was similar under both storage temperature regimes. However, apples treated with 1-MCP and stored at 3 °C softened less than those stored at 5 °C over the first four months of storage,

but after January fruit firmness declined sharply in all treatments, except at 3 ºC with 1-MCP.

Does storage in less stringent CA conditions compromise texture, flavour and fruit acid composition of low ethylene apples?The use of low-stringency CA storage was also investigated as a method of maintaining texture and flavour profiles during storage. Of particular interest was the effect of storage environment on fruit acidity profiles. In 2005/06 and 2006/07, ‘E55/55’, ‘E19/13’ and ‘Gloster 69’ were stored in either 21% O2 (air), 5% O2, 3% O2 and 2% O2 at 1.5 °C. Fruits were removed from storage in January, March and May 2007.

Storage in 2% oxygen provided the firmest fruit for all apple lines (Figure 6). ‘E19/13’ was the only apple line where storage in 3% oxygen or 5% oxygen reduced softening after six months storage. In ‘E55/55’ apples, only storage in 2% oxygen had any benefit over air storage and in ‘Gloster 69’, storage in 3% and 5% oxygen reduced fruit softening for the first four months of storage but the effect was lost after six months storage. Increasing the concentration of oxygen resulted in a decline in firmness for ‘E55/55’ and ‘Gloster 69’ apples. ‘E19/13’ apples only softened significantly when stored in air. The firmness of ‘E55/55’ and ‘Gloster 69’ apples declined from January to March, ‘Gloster 69’ softened the most and by March only apples stored in 2% oxygen retained an acceptable level of firmness. Measurements

SID 5 (Rev. 3/06) Page 13 of 20

Table 1. Concentration of malic acid (mg ml-1) in low ethylene apple lines stored under different concentrations of oxygen at 1.5 °C in 2006/07. Measurements were the mean of 2 replicate samples of apple; each replicate was the sum of 10 fruits. LSD0.05 = 0.625 on 39 df.

Apple line % Oxygen Malic acid concentration (µg ml-1)Harvest Jan March May

‘E19/13’ 21 6.7 5.77 5.07 2.985 5.36 5.77 2.703 5.51 5.81 1.772 5.43 5.42 1.76

‘E55/55’ 21 3.30 2.16 2.19 1.195 2.54 1.92 1.323 2.43 2.43 1.022 2.43 2.12 1.70

‘Gloster 69’ 21 9.62 3.34 3.94 2.315 4.00 3.70 1.323 4.15 3.60 3.712 3.77 3.21 2.29

of fruit acidity for the 2005/06 season identified malic acid as the major component of fruit acids with smaller amounts of oxalic acid (Annex 1, Tables 4&5). The concentration of acids was significantly different between varieties and in general, concentrations declined during storage. However, there was no significant effect of oxygen concentration on malic or oxalic acid concentrations. No physiological disorders of fruits under different storage oxygen regimes were detected.

In 2006/07, concentrations of malic acid varied considerably between ‘E19/13’, ‘E55/55’ and ‘Gloster 69’ at harvest (Table 1) and were generally lower than the previous year. The concentration of malic acid fell during storage and lowering the concentration of oxygen in the storage environment didn’t prevent its decline. Ascorbic acid rather than oxalic acid was detected as the second most prominent acid component in apple juice in 2006/07. Interestingly, ascorbic acid was not detected in harvest samples and the concentration in samples taken during storage varied (Annex 1, Table 6). Reducing the concentration of oxygen in the storage environment did not effect the overall concentration of ascorbic acid, other than in ‘E19/13’ apples where storage in 2% or 3% oxygen had lower ascorbic acid concentrations than those stored at higher oxygen concentrations.

The concentration of fructose in apple juice was generally lower in ‘E19/13’ apples than ‘E55/55’ and ‘Gloster 69’ (Annex 1, Table 7). Fructose concentrations in the three lines didn’t change significantly with inspection date or with the concentration of storage oxygen. Glucose concentrations of ‘E19/13’ and ‘E55/55’ apples were lower than in ‘Gloster 69’ apples. In general, glucose concentration increased in fruit sampled in January, March and May compared to harvest samples, but there was no effect of storage oxygen on glucose concentration (Annex 1, Table 8). In contrast, sucrose concentrations in all three apple lines rose during the early stages of storage before declining in March and May (Annex 1, Table 9). ‘E19/13’ apples had higher sucrose concentration than ‘E55/55’ and ‘Gloster 69’ fruit.

Both the concentration and the number of flavour volatiles detected in the low ethylene lines were greatly reduced compared to ‘Fiesta’. Amyl, butyl, and hexyl acetates make an important contribution to apple flavour and concentrations of these volatiles measured in the head-space above ‘Gloster 69’, ‘E55/55’ and ‘E19/13’ tissue were greatly reduced compared to values measured in ‘Fiesta’ (Table 2). Volatile concentrations were also divided by published ‘odour threshold of detection concentrations’ to give 'odour units'. When expressed in these terms, the volatile profile of the low ethylene lines was fairly similar to ‘Fiesta’ but the odour units were much lower (Annex 1, Table 10).

Do apples with a naturally low capacity to produce ethylene respond to treatment with 1-MCP during air storage at 3.5°C and are there adverse effects on the development of physiological disorders during storage? Late maturing progeny were on average 4 N firmer than earlier maturing fruit and treatment with 1-MCP generally increased the firmness by 20 N across all four categories (Figure 7). Of particular interest were the early maturing ‘D111’, ‘D113’ and ‘D157’ apple lines, these maintained good ex-store firmness after five months of storage in air of 76 N, 79 N and 75 N, respectively, without the aid of 1-MCP. These particular progeny may facilitate the development of apple lines that have many of the attributes

SID 5 (Rev. 3/06) Page 14 of 20

Table 2. The production of iso-amyl, butyl and hexyl acetates in ‘Fiesta’ and three ‘low ethylene’ producing lines. Flavour volatile concentration was determined using a purge-and-trap headspace technique, and then by automated thermal desorption (ATD) linked GC-MS.

Apple line Volatile concentration (ppb)Iso-amyl acetate

Butyl acetate Hexyl acetate

‘Fiesta’ 130 4000 1000‘Gloster 69’ 180 720 840‘E55/55’ 17 400 ND‘E19/13’ 23 48 180ND = not detected

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Figure 7. Effects of 1-MCP on firmness of ‘Gloster 69’ x ‘Fiesta’ (D-family) progeny. Values are the mean of two replicates per treatment; each replicate was the sum of ten fruits, with two measurements of firmness per fruit. LSD0.05 = 4.72 on 74 df.

of moderately high ethylene producing apples such full apple flavour and aroma combined with good texture and long storage life.

There was no significant interaction between 1-MCP and ethylene production capacity of D-family fruits. Some progeny were susceptible to ‘chemical burning’ of russetted areas of the fruit by 1-MCP and also brown flecks occurred on the surface of the skin in other apple lines after treatment (Annex 1, Figure 1). The incidence of rotting in ‘D-family’ apples was generally higher in late-producing ethylene apple lines (Annex 1, Figure 2) although application of 1-MCP increased the severity of rotting in some members of the progeny.

DiscussionThe potential to use low stringency storage (in air at higher than recommended temperatures) and apple lines with a heritable trait for low ethylene production as alternatives to CA storage to maintain fruit texture and flavour was determined. In terms of firmness, storage at 1.5-2.0 °C or 5 °C increased the storage period for ‘E19/13’ and ‘E55/55’ apples by 6-8 weeks compared to storage in air at 3.5 °C. The rate of softening of ‘E19/13’ and ‘E55/55’ apples were similar in 2004/05 and 2006/07 despite a 3-3.5 °C difference in storage temperatures while apples from 2005/06 stored at 3-3.5 °C softened to a greater extent. This suggests that seasonal factors may be more influential than storage temperature in maintaining firmness. Nevertheless, the low ethylene lines have the potential to be stored in air at higher storage temperatures (5 °C) without unacceptable losses in firmness. No physiological disorders were recorded in any of the apple lines during or after storage.

Application of 1-MCP further increased the storage life of ‘E19/13’, ‘E55/55’ and ‘Gloster 69’ apples. This approach could provide opportunities to store fruit at higher temperatures for extended periods without reductions in quality, thereby reducing energy inputs and the need for expensive CA technology. Moreover, the beneficial effect of 1-MCP on the storage life of low ethylene apples provides further evidence that the mechanism responsible for low ethylene production is linked to the fruits’ ability to synthesise ethylene rather than as a result of reduced ethylene perception.

At the outset, it was realised that prolonged air storage would decrease fruit acid concentrations and so experiments were carried out to determine the concentration of oxygen needed to balance the loss in acid levels with the maintenance of firmness and flavour volatile production during storage. Concentrations of malic acid, the primary organic acid in apple fruit, decreased during air storage in ‘Gloster 69’, ‘E55/55’ and ‘E19/13’ and these declines could not be prevented by lowering the oxygen concentration in store. Concentrations of oxalic and ascorbic acid also declined during storage and there were no significant effects of oxygen concentration on oxalic acid concentrations. Low oxygen concentrations (2% and 3%) reduced concentrations of ascorbic acid.

Apart from contributing to overall acidity, fruit acids also act as co-factors, reducing agents or substrates for enzymic reactions. Malic acid acts as a major substrate for respiration and is more readily metabolised than ascorbic acid or oxalic acid. The reduced form of ascorbic acid, dehydroascorbic acid, can react with amino acid side chains on cell wall polypeptides and prevent cross linking of matrix polysaccharide (Smirnoff, 1996). Moreover, oxalic acid can also contribute to cell wall loosening by interacting with divalent copper and calcium ions in the cell wall. The utilisation of

SID 5 (Rev. 3/06) Page 15 of 20

acids in fruits during storage may not only impact on the taste of fruits but also contribute to a decline in texture during storage.

The sugar profiles in the different apple varieties during storage demonstrated that ‘E19/13’ apples had lower fructose and glucose, but higher sucrose concentrations than higher ethylene producing ‘E55/55’ and ‘Gloster 69’ apples. Since the breakdown of sucrose into its constituent fructose and glucose molecules by invertase is ethylene-dependant, the breakdown of sucrose in ‘E19/13’ apples was likely to be limited. The ratio of fructose, glucose and sucrose is important and can affect consumer perception of eating quality.

The presence and concentration of flavour volatiles also make an important contribution to organoleptic quality in apple. Since the production of flavour volatiles seems to be linked to ethylene perception and/or ethylene action, we tested whether volatile production was affected by the low ethylene trait. GC-MS analyses indicated that the production rates of butyl, amyl and hexyl acetates, which contribute to apple flavour (Baldwin, 2002), were impaired in the low ethylene lines compared to those measured in ‘Fiesta’. Volatiles were also ranked in terms of ‘odour units’ which can be used to estimates the importance of their contribution to overall taste. Although the profile of the top seven volatiles ranked in this was similar in the four apple lines, the odour unit values were reduced by up to 74% in the low ethylene lines. A thorough analysis of fruit organoleptic quality in the progenies raised will form an important component of any follow-on project to improve the sustainability of UK top fruit storage.

Some of the progeny from the D-family were susceptible to skin damage after treatment with 1-MCP and this suggests that there may be a genetic predisposition of apples to 1-MCP skin damage. Of further interest was the high degree of ‘core flush’ observed in ‘D64’ (50%), ‘D78’ (70%) and ‘D174’ (100%) apple lines suggesting a genetic predisposition to developing the disorder during storage. These progeny may be a useful resource in our future attempts to identify a genetic marker for the propensity to develop core flush during storage. The incidence of rotting in D-family apples was generally higher in low ethylene lines and treatment with 1-MCP exacerbated this problem in some lines. Ethylene is known to trigger host defence mechanisms in plants and so it is likely that fruits with an inherently low capacity to synthesise ethylene may be less likely to prevent colonisation by pathogenic fungi.

Objective 4IntroductionTwo individuals from the 'E55' and 'E19' progenies, 'E55/55' and 'E19/13', showed maximum ethylene production rates of 3% and 0.5%, respectively, of the 'Fiesta' parent (Defra HH2606STF). Furthermore, production rates of 'E55/55' and 'E19/13' were very much lower than 'Gloster 69'. In HH2606STF, we determined that the low ethylene trait could be partly explained by a mutated, ripening-specific ACC synthase (ACS) gene (MdACS-1). 'Fiesta' is heterozygous for MdACS-1 while 'Gloster 69', 'E55/55' and 'E19/13' are homozygous for this mutated gene. Therefore, another, as yet unknown gene must underlie the different ethylene production rates of 'Gloster 69' and 'E55/55' and 'E19/13'. We used bulk segregant analysis (BSA) (Mitchelmore et al., 1991) to try to identify additional markers linked to other component(s) of the low ethylene trait.

Materials and MethodsBulk segregant analysis was performed on genomic DNA extracted from the D-family progeny (‘Fiesta’ × ‘Gloster 69’) which take different lengths of time to synthesise a threshold ethylene production rate of 1 nL g-1 h-1 (Defra HH2606STF). DNA samples were pooled from eight early (< 15 days), eight mid-early (58-91 days), eight mid-late (159-210 days) and eight late (> 250 days) ethylene producing apple lines. The bulks were screened against 86 pairs of microsatellite primers and amplicons generated sized with 500 LIZ™ in an ABI 3100 Genetic Analyzer running GENESCANâ 3.7 and GENOTYPERâ 3.7 software (Applied Biosystems). Putative markers linked to late ethylene producers were identified and screened in the full progeny. The gene-specific markers MdACS-1 and MdETR-1 were also screened in all the seedlings.

Gene-specific PCR markers for ACO and ACS were passed to the East Malling Apple & Pear Breeding Club where they were used to determine the genotypes of parental material.

ResultsA bulk segregant analysis approach was used to discriminate between the D-family apple lines. SSR markers, CH05C06 and CH05E04, mapping to LG16, amplified differential peaks in the bulks and were

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Figure 8. A map constructed from 136 seedlings from the cross ‘Fiesta’ × ‘Gloster 69’ (distances in cM) with schematics showing Percentage variance explained (red) and LOD scores (blue) for a major low ethylene QTL on LG15 near a mutated, ripening-specific ACC synthase (ACS) gene (MdACS1-1).

putatively linked to the phenotype. CH02A03 and CH01F03a, closely linked to the two markers on the ‘Fiesta’ × ‘Totem’ map (Fernández-Fernández et al. 2008), were also tested on each of the 32 D-family progeny making up the bulk samples before the whole progeny was screened. The markers were mapped using JOINMAP® 4 and QTL analysis for late ethylene production (days to 1 nL g-1 h-1) was performed using MapQTL® 5 but none was found (data not shown). The gene markers MdACS-1 and MdETR-1 were screened with 136 D-family seedlings along with six SSRs (CH01d08, CHO2c09, CHO2d11, CHO3b10, CHO3h06 and CH04g10) all mapping to LG15 in ‘Fiesta’ × ‘Totem’ (Fernández-Fernández et al. 2008). The same strategy found a major QTL for late ethylene (Figure 8) spanning MdACS-1, MdETR-1 and three SSRs - CH02d11, CH03b06 and CH03h03. MdACS-1 explained most of the 70% variance. Heterozygotes for MdACS-1, segregating in ‘Fiesta’ only, were removed from the dataset and the late ethylene data re-analysed to look for ‘non ACS1-1’ QTLs. A second QTL was identified on LG15 (Figure 9) explaining 15% of the variance.

DiscussionAlthough a reduced capacity to produce ethylene can help to maintain firmness under low stringency storage conditions, our results suggest that very low concentrations of ethylene can impair flavour

volatile production and predispose fruit to physiological disorders. Therefore, it will be important to distinguish between ‘low’ and ‘very low’ ethylene producing lines in our future efforts to improve the storage potential of apples. We have already determined that 'E55/55' and 'E19/13' apples showed maximum ethylene production rates of 3% and 0.5%, respectively, of the 'Fiesta' parent and production rates of 'E55/55' and 'E19/13' were very much lower than 'Gloster 69’ (Defra HH2606STF). We determined that most of the 70% of the variance for the low ethylene trait could be explained by a mutated, ripening-specific ACC synthase (ACS) gene (MdACS-1). 'Fiesta' is heterozygous for MdACS-1 while 'Gloster 69',

'E55/55' and 'E19/13' are homozygous for this mutated gene. Therefore, another, as yet unknown gene must underlie the different ethylene production rates of ‘Gloster 69' and 'E55/55' and 'E19/13'. A ‘non ACS-1’ QTL explaining 15% of the variance was also found on LG15. Three SSRs (CH02d11, CH03b06 and CH03h03), heterozygous in both D-family parents, mapped to the region explaining 15% of the variance for the trait and could be candidates for use in future marker-assisted selections.

Objective 5IntroductionThere are many opportunities using conventional plant breeding to develop new apple cultivars that have improved storage characteristics. EMR has available a range of accessions with useful storage traits in its genebank and has acquired others from the National Fruit Collections, Brogdale. These include ‘Juliet’ (‘COOP 44’) and SA544-48 which maintain firmness and crispness in cold storage for over one year, and ‘Hambledon Deux Ans’ which is said to be barn storable for two years but suffers from low temperature breakdown in cold storage (Alston, 1988). In addition, there are several cultivars which are unduly susceptible to storage disorders including ‘Beauty of Bath’, ‘Egremont Russet’ and

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‘McIntosh’ which are prone to mealiness, bitter pit and core flush, respectively. Four progenies were screened for resistance to storage disorders by Alston (1988) and several of the more promising parental lines are still available at EMR. These, and other, cultivars were used in crosses to raise new progenies that segregate for the afore-mentioned storage characters.

Materials and MethodsA range of crosses was made in 2004 among nine different varieties with differing storage capacities. The varieties used were ‘Beauty of Bath’ and ‘Lady Sudeley’ (both with minimum keeping quality), ‘Egremont Russet’ (susceptible to bitter pit), ‘McIntosh’ (susceptible to core flush), ‘Juliet’ (a long storing apple from the USA), ‘Jester’ (carries genes for susceptibility to senescent breakdown and core flush), ‘Hambledon deux Ans’ (reputed to ‘barn store’ for 2 years) and ‘SA544-48’ (an East Malling selection that is still firm after 1 year in cold storage). Several varieties were crossed with ‘Fiesta’ to enable reference to the established apple genome map. Seeds from five of the crosses were sown in November 2004 (E810 – ‘Lady Sudeley’ × ‘Hambledon deux Ans’, E811 - ‘Lady Sudeley’ × ‘Juliet’, E830 - ‘Cox’ × ‘Jester’; E831 – ‘Fiesta’ × ‘Beauty of Bath’, E832 – ‘Fiesta’ × ‘Egremont Russet’, E833 – ‘McIntosh’ × ‘Fiesta’). Once germinated, the seedlings were potted up ready for planting out in early summer 2005.

Some of the original crosses made in 2004 produced too few seeds to justify planting so these crosses were repeated in 2005 to complete the set. Seeds from three of the crosses were sown in November 2005 (E849 – ‘Fiesta’ × ‘Lady Sudeley’, M520 – ‘SA544-48’ × ‘Lady Sudeley’ and M519 – ‘SA544-48’ × ‘Beauty of Bath’). One cross (‘Lady Sudeley’ × ‘Hambledon deux Ans’) failed completely for the second year indicating full incompatibility. Once germinated, the seedlings were potted up ready for planting out in early summer 2006.

ResultsThe following seedlings from the progenies created in 2004 were planted in the field in a sprayed plot: ‘E811’ (‘Lady Sudeley’ × ‘Juliet’, 180 seedlings); ‘E830’ (‘Cox’ × ‘Jester’, 188 seedlings); ‘E831’ (‘Fiesta’ × ‘Beauty of Bath’, 181 seedlings); ‘E832’ (‘Fiesta’ × ‘Egremont Russet’, 180 seedlings) and ‘E833’ (‘Wijcik’ × ‘Fiesta’, 198 seedlings).

Ten trees of each of ‘E19/13’, ‘E55/55’ and ‘Gloster 69’ were propagated onto ‘M.9’ rootstocks in February 2005. These trees were planted in the field, together with trees of ‘Juliet’, to provide fruit for a future project. Two hundred seedlings from the cross E849 – ‘Fiesta’ × ‘Lady Sudeley’ (minimum keeping quality) - were planted out in early summer 2006.

The surviving seedlings from the progenies created in 2004 were bench-grafted onto ‘M9’ rootstocks in January 2007: ‘E811’ (‘Lady Sudeley’ × ‘Juliet’, 180 seedlings); ‘E830’ (‘Cox’ × ‘Jester’, 188 seedlings) and ‘E832’ (‘Fiesta’ × ‘Egremont Russet’, 180 seedlings).

DiscussionApples with improved storage characteristics will eventually facilitate the use of less stringent, more sustainable storage regimes that optimise fruit quality. Three progenies were raised that will produce fruit of varying keeping qualities for assessment in a future project and thus enable the mapping and marker development for these key traits. The progenies ‘E811’, ‘E830’ and ‘E832’ will facilitate future work aimed at improving the sustainability of storage regimes for UK produced top fruit. EMR is currently evaluating how best to exploit these unique genetic resources.

Objective 6The technology and knowledge transfer activities undertaken by the project team throughout the duration of the project are listed under ‘Knowledge Transfer’ (Page 18).

At the outset, it was envisaged that the storage behaviour of fruit from the new progenies with natural traits for improved storage raised under Objective 5 would be determined in a follow-on Horticulture LINK project. In such a project, the physical, chemical, and molecular basis of fruit quality were to be combined with sensory evaluations to characterise the storage potential of the new progenies. The storage performance (flavour development, textural changes and susceptibility to physiological disorders and pathological diseases) of fruit picked at different stages of maturity was to be determined under a range of temperatures and concentrations of oxygen and carbon dioxide. This information would be useful to breeders at EMR to increase the portfolio of useful markers for future marker-assisted selection.

The feasibility of developing a Horticulture LINK proposal that would build upon the results and genetic resources developed during the current project was discussed with industry representatives

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several times during the final two years of the project. The industry was, and remains, very concerned about the more pressing problem of Diffuse Browning Disorder (DBD) and did not view storage potential as a research priority area. This lack of industry support, coupled with EMR’s enforced shift away from top fruit research and the re-alignment of Defra Policy Areas, mean that it was no longer feasible to consider developing a Horticulture LINK proposal. EMR is currently evaluating how best to exploit the unique genetic resources developed in this project.

Main implications of the findingsMarkers developed for softening-related genes are likely to be valuable for breeding apple lines with enhanced fruit texture. Although a reduced capacity to produce ethylene can help to maintain firmness under low stringency storage conditions, our results suggest that very low concentrations of ethylene can impair flavour volatile production and predispose fruit to physiological disorders. Therefore, apple lines with low, rather than very low, rates of ethylene production offer the best opportunity to reduce the stringency of CA regimes while maintaining full apple flavour and aroma, good texture and long storage life. Markers for the low ethylene trait will help to discriminate between low ethylene producing lines in future breeding work to improve texture and flavour attributes of stored apples while reducing the costs and energy inputs associated with CA storage.

Future workDiscussions will be held with key industry representatives during 2008 to determine whether improving storage potential and reducing the costs and inputs associated with high stringency CA regimes are likely to be a priority in the short-term.

Knowledge transferPublicationsColgan R, Stow J, Marchese A, Nidzovic S, Else MA (2006). Storage quality of low ethylene producing

apples. Journal of Fruit and Ornamental Plant Research 14, 85-91.Optimising texture and flavour in stored apples. Plant It! October 2004, Issue 6, p 3-4.

Meeting ProceedingsRichard Colgan, Sladjana Nidzovic, Annalisa Marchese, Mark Else (2004). The mechanism for low

ethylene production in apple (Malus pumila). Advances in Applied Biology: Providing new opportunities for consumers and producers in the 21st century,. 15-17 December 2004, St Catherine's College, Oxford, UK.

Richard Colgan, Sladjana Nidzovic, Annalisa Marchese, John Stow, Mark Else (2005). Storage Quality of low ethylene producing apples. EUFRIN Workshop on ‘Methods and legal regulation in fruit quality determination’; June 16-18, 2005, Institute of Pomology and Floriculture, Skierniewice, Poland.

PresentationsOptimising texture and flavour in stored apples – an overview. Presentation at EMRA Members’

Storage Day, March 2004.Apple Research art EMR (2004). Exhibit at ‘The Living Land’ – Kent Schools’ Farm Fair, Kent, April

2004.Regulations in Fruit Quality Determination’ workshop held at the Research Institute of Pomology and

Floriculture, Skiernievice, Poland, June, 2005.‘What alternatives to cold storage exist and how do the impacts of these alternatives compare with cold

storage?’ Presented at a seminar to inform the work of the Food Climate Research Network ‘Exploring the impact of the sector on the UK’s greenhouse gas emissions and the options for achieving emissions reductions’. Manchester Business School, December 2005.

How can a banana make an apple ripen? Interactive exhibit at EMR’s Science, Engineering and Technology Week event, March 2005.

Talk given to the West Sussex Fruit Group, July 2006.Talk given to the RHS Fruit Group, September 2006.‘Storage Quality of Low Ethylene Producing Apples’. Presented at the ‘Methods and Legal ripening – a

current understanding. Presentation at the EMRA Members' Day, March, 2007.The use of Real-Time PCR in gene expression studies of ripening apple. Talk given to the Kent

Ambassadors’ visit to EMR, August, 2007.

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CA storage of apples and control of ethylene. Talk given to the Kent Ambassadors’ visit to EMR, August, 2007.

Two posters presented at the fruit section of Eucarpia in Zaragoza, September 2007.Talks to the public on apple breeding at EMR given at the at the EMR ‘Apple Day’ weekend,

September 2007.

Meetings attendedMethods and Legal Regulations in Fruit Quality Determination’, Research Institute of Pomology and

Floriculture, Skiernievice, Poland, June 2005.Exploring the impact of the sector on the UK’s greenhouse gas emissions and the options for achieving

emissions reductions. Manchester Business School, December 2005.Fruit Logistica, Berlin, February, 2006.Fruit Logistica, Berlin, February, 2007.Symposium of the fruit section of Eucarpia in Zaragoza, September 2007.

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

Alston FH (1988). Breeding apples for long storage. Acta Horticulturae (Fruit Breeding), 224, 109-117.

Baldwin E (2002). Fruit flavour, volatile metabolism and consumer perception. In: Fruit Quality and is Biological Basis. Ed. Michael Knee, pp 89.

Bauchot AD, Mottram DS, Dodson AT, John, P (1998). Effect of aminocyclopropane-1carboxylicacid oxidase antisense gene on the formation of volatile esters in Cantaloupe Charentais melon (Cv. Védrandais). Journal of Agriculture, Food and Chemistry 46, 4787-4792.

Brown DS, Buchanan JR, Hicks JR (1966). Volatiles from apple fruits as related to variety, maturity and ripeness. Proceedings of the American.Society for Horticultural Science 88, 98-108.

Fan X, Mattheis JP, Buchanan D (1998). Continuous requirement of ethylene for apple fruit volatile synthesis. Journal of Agriculture, Food and Chemistry 46, 1959-1963.

Fernández-Fernández F, Evans KM, Clarke JB, Govan CL, James CM, Marić S, Tobutt KR (2008). Development of an STS map of an interspecific progeny of Malus. Tree Genetics and Genomes DOI: 10.1007/s11295-007-0124-y

Fulton TM, Grandillo S, Beck-Bunn T, Fridman E, Frampton A, Lopez J, Petiard V, Uhlig J, Zamir D, Tanksley SD (2000). Advanced backcross QTL analysis of a Lycopersicon esculentum × Lycopersicon parviflorum cross. Theoretical and Applied Genetics 100, 1025-1042

King GJ, Maliepaard C, Lynn JR, Alston FH, Durel CE, Evans KM, Griffon B, Laurens F, Manganaris AG, Schrevens E, Tartarini S (2000). Quantitative genetic analysis and comparison of physical and sensory descriptors relating to flesh firmness in apple (Malus pumila Mill.) Theoretical and Applied Genetics 100, 1074-1084.

Michelmore R, Paran I, Keselli V (1991). Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proceedings of the National Academy of Science USA 88, 9828-9832.

Seymour G, Poole M, Manning K, King G. (2008). Genetics and epigenetics of fruit development and ripening. Current Opinions in Plant Biology 11, 58-63.

Shakespeare, L (2001). The Biochemistry of Apple Fruit Softening. Ph.D. Thesis p159.Smirnoff N. (1996). The function and metabolism of ascorbic acid in plants, Annals of Botan, 78,

661-669.Van Ooijen JW, Voorrips RE (2001) JoinMap® 3.0, Software for the calculation of genetic

linkage maps. Plant Research International, Wageningen, the NetherlandsYuan JS, Reed A, Chen F, Stewart CN (2006). Statistical analysis of real-time PCR data (2006)

BMC Bioinformatics 7, 85

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