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Bespuitingen met de bladmeststof ATS zijn al meer dan tien jaar een door veel fruittelers toegepaste teelthandeling om de vruchtzetting bij appel te beperken. Vooral bij uitbundig bloeiende bomen kunnen telers bij een juist toepassingsmoment de vruchtzetting verkleinen. Later is dan minder chemische vruchtdunning en arbeidsintensieve handdunning nodig. Recent onderzoek van WUR/PPO Fruit wijst uit dat de huidige wijze van vruchtdunning aangepast dient te worden.

De belangrijkste appelrassen van dit moment (Elstar, Junami, Kanzi, Rubens) hebben gemeen dat zij allemaal regelmatig gedund moeten worden om het juiste drachtniveau te verkrijgen, de gewenste vruchtmaat en vruchtkwaliteit (maat, suikergehalte) te behalen en om beurtjaren in de productie te vermijden. Handdunnen is arbeidsintensief en dus kostbaar. Bovendien vindt handdunnen uit praktische overwegingen pas plaats na de junirui, wat te laat is om beurtjaren te voorkomen. Een betrouwbare chemische vruchtdunning betekent een grote besparing op arbeidskosten voor dunnen en geeft de beste garantie op het vermijden van beurtjaren. Met de thans gebruikte dunmiddelen ATS en MaxCel lukt het niet om onder alle omstandigheden en bij elk ras de gewenste mate van dunning te behalen. Weersomstandigheden, het bloeistadium dan wel het ontwikkelingsstadium van de jonge vruchtjes, het groeiniveau van de boom en verschillen tussen de takken binnen de boom spelen een belangrijke rol bij de mate van dunnende werking.

De belangrijkste appelrassen van dit moment (Elstar, Junami, Kanzi, Rubens) hebben gemeen dat zij allemaal regelmatig gedund moeten worden om het juiste drachtniveau te verkrijgen, de gewenste vruchtmaat en vruchtkwaliteit (maat, suikergehalte) te behalen en om beurtjaren in de productie te vermijden. Handdunnen is arbeidsintensief en dus kostbaar. Bovendien vindt handdunnen uit praktische overwegingen pas plaats na de junirui, wat te laat is om beurtjaren te voorkomen. Een betrouwbare chemische vruchtdunning betekent een grote besparing op arbeidskosten voor dunnen en geeft de beste garantie op het vermijden van beurtjaren. Met de thans gebruikte dunmiddelen ATS en MaxCel lukt het niet om onder alle omstandigheden en bij elk ras de gewenste mate van dunning te behalen. Weersomstandigheden, het bloeistadium dan wel het ontwikkelingsstadium van de jonge vruchtjes, het groeiniveau van de boom en verschillen tussen de takken binnen de boom spelen een belangrijke rol bij de mate van dunnende werking.

Traditionele veredeling is bij fruitbomen enorm tijdrovend. Veredeling met behulp van genetische modificatie kan het proces erg versnellen, maar dit stuit in Europa op veel maatschappelijke weerstand. Appelveredelaar Henk Schouten van PRI denkt met cisgenese alleen de voordelen van genetische modificatie te introduceren in de traditionele appelveredeling.

Bespuitingen met de bladmeststof ATS zijn al meer dan tien jaar een door veel fruittelers toegepaste teelthandeling om de vruchtzetting bij appel te beperken. Vooral bij uitbundig bloeiende bomen kunnen telers bij een juist toepassingsmoment de vruchtzetting verkleinen. Later is dan minder chemische vruchtdunning en arbeidsintensieve handdunning nodig. Recent onderzoek van WUR/PPO Fruit wijst uit dat de huidige wijze van vruchtdunning aangepast dient te worden.

In een orienterende proef werd gedurende vier jaar in Randwijk de groei, productie en vruchtkwaliteit van ‘Elstar’ op de onderstammen G.11, G.41, P 59, P 60, P 66 en P67 vergeleken met die op de standaardonderstam M.9 T337. Na 4 jaar was de cumulatieve productie en het gemiddeld vruchtgewicht van ‘Elstar’ met uitzondering van P 67 op deze onderstammen vergelijkbaar tot iets hoger dan op M.9 T337. De bladstand en productiviteit van de bomen op P 67 verslechterde sterk gedurende de duur van de proef, waarschijnlijk ten gevolgde van een of andere ziekte. G.11 en G.41 hadden ten opzichte van de overige onderstammen en M.9 T337 het pluspunt dat zij een zeer gladde vergroeiing vertoonden met ‘Elstar’ en geen wortelvelden vertoonden. Daarnaast gaf G.41 bij een gemiddeld iets hogere productie een hoger aandeel goed gekleurde vruchten (>33% blos) bij een vergelijkbare goede vruchtmaat als bij M.9 T337.

In een orienterende proef werd gedurende vier jaar in Randwijk de groei, productie en vruchtkwaliteit van ‘Elstar’ op de onderstammen G.11, G.41, P 59, P 60, P 66 en P67 vergeleken met die op de standaardonderstam M.9 T337. Na 4 jaar was de cumulatieve productie en het gemiddeld vruchtgewicht van ‘Elstar’ met uitzondering van P 67 op deze onderstammen vergelijkbaar tot iets hoger dan op M.9 T337. De bladstand en productiviteit van de bomen op P 67 verslechterde sterk gedurende de duur van de proef, waarschijnlijk ten gevolgde van een of andere ziekte. G.11 en G.41 hadden ten opzichte van de overige onderstammen en M.9 T337 het pluspunt dat zij een zeer gladde vergroeiing vertoonden met ‘Elstar’ en geen wortelvelden vertoonden. Daarnaast gaf G.41 bij een gemiddeld iets hogere productie een hoger aandeel goed gekleurde vruchten (>33% blos) bij een vergelijkbare goede vruchtmaat als bij M.9 T337.

Bespuitingen met de bladmeststof ATS zijn al meer dan tien jaar een door veel fruittelers toegepaste teelthandeling om de vruchtzetting bij appel te beperken. Vooral bij uitbundig bloeiende bomen kunnen telers bij een juist toepassingsmoment de vruchtzetting verkleinen. Later is dan minder chemische vruchtdunning en arbeidsintensieve handdunning nodig. Recent onderzoek van WUR/PPO Fruit wijst uit dat de huidige wijze van vruchtdunning aangepast dient te worden.

Effectiviteit bepalen van electrolysewater ter bestrijding van schurft, Venturia inaequalis op appelbladeren.

The aim of this thesis was finding genes that control the production of potentially health beneficial metabolites in apple fruits. The approach was genetic mapping of secondary metabolites such as phenolic compounds in an F1 progeny, leading to the detection of genetic loci that controlled these metabolites. At these genetic loci candidate genes were identified, using the whole genome sequence of apple, and it was investigated whether the expression of these candidate genes in the F1 progeny correlated with the metabolite levels. The cultivated apple (Malus x domestica Borkh) is among the most diverse and ubiquitously cultivated fruit species. It belongs to the family of Rosaceae which includes many commercial fruit species such as pear, strawberry, cherry, peach, apricot, almond, black cherry, and crab apple. Apple has a haploid chromosome number of 17. It is a self-incompatible and highly heterozygous crop. The breeding is further hampered by the long juvenile period which makes breeding in this crop a very slow process. The saying “An apple a day keeps the doctor away” has encouraged many researchers to search for the “magic” ingredients found in apple. Due to the beneficial role of apple phenolics, it is also called as a “new agrochemical crop”. Apple possesses many health beneficial properties for human beings as it is a rich source of phenolic compounds.It has been associated with reducing the risks of certain diseases such as cancers, particularly prostate, liver, colon, and lung cancers, cardiovascular diseases, coronary heart diseases, asthma, type-2 diabetes, thrombotic stroke, and ischemic heart disease. The second chapter of this thesis describes the construction of genetic linkage maps of the parents of a segregating population derived from the cross between the cultivars ‘Prima’ and ‘Fiesta’. For this purpose the already available linkage maps, as described in this chapter, were made denser by inclusion of 240 Diversity Array Technology (DArT) markers. Thus the total number of markers for ‘Prima’ and ‘Fiesta’ integrated map reached to 820. DArT-markers are hybridization based dominant DNA-markers. DArT provides a high-throughput whole genome genotyping platform for the detection and scoring of hundreds of polymorphic loci without any need for prior sequence information. This is the first report on DArT in horticultural trees. Genetic mapping of DArT markers in two mapping populations and their integration with other marker types showed that DArT is a powerful high throughput method for obtaining accurate and reproducible marker data, at low cost per data point. This method appears to be suitable for aligning the genetic maps of different segregating populations. Sequencing of the marker clones showed that they are significantly enriched for low copy, gene rich regions. Chapter 3 describes metabolic diversity of Malus. Wild germplasm was compared to advanced breeding selections and to the segregating F1 population from the cross between the cultivars ‘Prima’ and ‘Fiesta’. The metabolic profiles were analyzed by means of liquid chromatography-mass spectrometry (LC-MS). LC-MS is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. This resulted in the detection of 418 putative metabolites in the peel and 254 in the flesh. Fruits from 23 wild species, eight advanced selections and the segregating F1 population were analyzed. The data were subjected to Principle Components Analysis (PCA). Variance analysis of the first PC showed that genetic variation accounted for 96.6 % in peel and 97.4 % in flesh of the total metabolic variation. Technical variation accounted for 1.4 % and 0.8%, while environmental variation accounted for 2.0% and 1.8% in peel and flesh respectively. The genetic variation between wild genotypes was very large, compared to the advanced selections and the F1 progeny. Only 8 % of the genetic variation of the first principle component was captured by the advanced selections. This indicates strong genetic erosion during breeding. This genetic erosion was mainly caused by reduction of the levels of several flavonoids including catechin, epicatechin and procyanidins. PCA of the F1 progeny of the ‘Prima’ x ‘Fiesta’ cross showed a clear 3:1 Mendelian segregation of metabolites. These metabolites were 4.2 fold less in both peel and flesh in progeny that had inherited the recessive alleles of a gene at the top of Linkage Group16 (LG16) from the heterozygous parents. We found a separate group of 11 metabolites in peel and 12 in flesh. These metabolites were putatively identified as glycosylated forms of b-glycols: R-octane-1, 3-diol and its unsaturated form R-5-(Z)-octene-1, 3-diol which have a potential role in controlling infection by microorganisms and influence the aroma of some ciders. The levels of these metabolites were up to 50 fold more abundant in some progeny compared to both parents. Genetic mapping showed that this strong increase was caused by one locus at the top of LG8, in progeny that had inherited only the recessive alleles of that locus from the heterozygous parents. This research illustrates not only the strong genetic erosion in apple breeding regarding metabolic diversity, and strong reduction of flavonoids in some progeny, but also shows that inbreeding can lead to a strong increase of metabolites that were present at much lower levels in both parents and advanced selections. This loss and gain of metabolites was especially observed in case of accumulation of recessive alleles during inbreeding. The genetic factors controlling metabolite composition were studied in more detail in Chapter 4. We investigated the genetic factors of the quantitative variation of these potentially beneficial compounds (Chapter 3, 4), by combining the genetic maps (Chapter 2) with the LC-MS data for thesegregating F1 population from the cross ‘Prima’ x ‘Fiesta’. This resulted into metabolite quantitative trait loci (mQTLs). When using the software MetaNetwork, 669 significant mQTLs were detected: 488 in the peel and 181 mQTLs in the flesh. Four linkage groups (LGs) i.e. LG1, LG8, LG13 and LG16 were found to contain mQTL hotspots, mainly regulating metabolites that belong to the phenylpropanoid pathway. These include various metabolites i.e. sinapate hexoside, coumaroyl hexoside, phloridzin, quinic acids, phenolic esters, kaempferol glycosides, quercetin glycosides, cyanidin pnetoside, flavan-3-ols (catechin, epicatechin), and procyanidins. The genetics of annotated metabolites was studied in more detail using MapQTL®. It was found that quercetin conjugates had mQTLs on LG1 and LG13. The most important mQTL hotspot with the largest number of metabolites was, however, detected at the top of LG16: mQTLs for 32 peel-related and 17 flesh-related phenolic compounds. The metabolites that mapped in the mQTL hotspot on LG16 all belong to the phenylpropanoid pathway of secondary metabolites. These compounds showed a monogenic Mendelian inheritance in a 3:1 segregation ratio. Procyanidins dimer II was used as a representative of the numerous compounds that mapped at the LG16 mQTL hotspot. By means of graphical genotyping of this monogenic trait, a genetic window could be made in which the gene that caused the mQTL hotspot should reside. We located structural genes involved in the phenolic biosynthetic pathway, using the genetic map together with the published whole genome sequence of apple. The structural gene leucoanthocyanidin reductase (MdLAR1) was detected in the mQTL hotspot window on LG16, as were seven transcription factor genes. To our knowledge, this is the first time that a QTL analysis was performed on such a high number of metabolites in an outbreeding plant species. The expression of the candidate genes found in the mQTL window on LG16 was studied and discussed in Chapter 5. qPCR was used for this purpose and it was found that the expression of only the structural gene MdLAR1 was strongly positively correlated with the metabolite procyanidin dimer II content. Neither the expression profiles of other structural genes of the phenylpropanoid pathway, the transcription factor genes at the mQTL hotspot, nor of transcription factor genes outside the mQTLs hotspot, showed any significant correlation with the procyanidin dimmer II content that mapped at the mQTL hotpot. This indicates that MdLAR1 was the gene, which caused this mQTL hotspot (Chapter 5). The progeny that had inherited one or two copies of the dominant alleles (Mm, MM) showed on the average a 4.4 and 11.8 fold higher expression level of MdLAR1 respectively, compared to the progeny that had inherited the recessive alleles only (mm). This led to a 4.0 fold increase of procyanidin dimer II level at the ripe stage. Strikingly, at the mQTL hotspot at the top of LG16, there is also a locus that controls acidity of the ripe fruits. However, the dominant alleles for acidity appeared to be in repulsion to the dominant alleles for high metabolite levels (Chapter 6). This shows that acidity is controlled by another gene than the metabolite levels. The combination of the genetic position based on the whole apple genome sequence, annotation of potential genes, and expression profiling indicated that the malic acid transporter gene MdALMT2 was responsible for the clear differences in malic acid content and pH in mature apple fruits of the segregating F1 population. The genetic inheritance of at least one dominant allele (MaMa/Mama) of this gene sufficed for a three-fold increase of the malic acid concentration and a reduction of the pH from 4 to 3 in ripe apples, compared to the presence of only the lower expressed recessive allele (mama). This malic acid transporter gene is located at the top of LG16.Malic acid is the predominant organic acid associated with the pH in apple fruits. It is synthesized in the cytoplasm and transported into the cell vacuole. The concentration of malic acid in the cell vacuole determines the pH of the cell. pH is very important for the overall taste of many fruits, including apple, and has profound effects on the organoleptic quality of apples. The pH of mature apples was genetically mapped on LG16 in the segregating population from the cross ‘Prima’ x ‘Fiesta’. To our knowledge, this is the first time that the genetic segregation of the pH in apple is assigned to a specific gene. Further, this gene has not been reported yet in conjunction to pH of apples or other fruits. After cloning of the MdALMT2 gene, it can be used for, proof of principle, influencing the acidic of existing varieties either by silencing this gene in more acidic cultivars or by inserting this gene into the low acidic cultivars. Another step would be to develop an allele specific molecular marker for selection (Marker Assisted Selection) of the acidity of fruits already at seedling stage, five years before the trees carry fruits. In another study, a dominantly mutated allele of the transcription factor gene MdMYB10,including its upstream promoter, coding region and terminator sequence, was introduced by transformation into apple, strawberry and potato plants. The dominantly inherited mutant allele of MdMYB10 from apple induces anthocyanin production throughout the plant, also at the early stage after transformation. The aim was to determine whether MdMYB10 could be used as a visible selectable marker for plant transformation as an alternative to chemically selectable markers, such as kanamycin resistance. After transformation, the color of calli, shoots and well-growing plants were evaluated. Red and green shoots were harvested from apple explants and examined for the presence of the MdMYB10 gene by PCR analysis. Red shoots of apple explants always contained the MdMYB10 gene but not all MdMYB10 containing shoots were red. Strawberry plants transformed with the MdMYB10 gene showed anthocyanin accumulation in leaves and roots. No visible accumulation of anthocyanin could be observed in potato plants grown in vitro, even the ones carrying the MdMYB10 gene. However, acid methanol extracts of potato shoots or roots carrying the MdMYB10 gene contained up to four times higher anthocyanin content than control plants. Therefore, anthocyanin production as a result of the dominant MdMYB1010 gene can be used as a selectable marker for apple, strawberry and potato transformation, replacing kanamycin resistance gene such as nptII. We reported this MdMYB10 as a cisgenic selectable marker gene for apple transformation (Chapter 7). The results from all experimental chapters have been discussed in a broader sense in the general discussion (Chapter 8). The future prospectives and potential challenges in the genetical metabolomics are also highlighted. The approaches we developed in the current thesis could be used not only for developing potentially a more healthy and improved apple but can also be applied for the genetical metabolomics studies in other important crops.

The aim of this thesis was finding genes that control the production of potentially health beneficial metabolites in apple fruits. The approach was genetic mapping of secondary metabolites such as phenolic compounds in an F1 progeny, leading to the detection of genetic loci that controlled these metabolites. At these genetic loci candidate genes were identified, using the whole genome sequence of apple, and it was investigated whether the expression of these candidate genes in the F1 progeny correlated with the metabolite levels. The cultivated apple (Malus x domestica Borkh) is among the most diverse and ubiquitously cultivated fruit species. It belongs to the family of Rosaceae which includes many commercial fruit species such as pear, strawberry, cherry, peach, apricot, almond, black cherry, and crab apple. Apple has a haploid chromosome number of 17. It is a self-incompatible and highly heterozygous crop. The breeding is further hampered by the long juvenile period which makes breeding in this crop a very slow process. The saying “An apple a day keeps the doctor away” has encouraged many researchers to search for the “magic” ingredients found in apple. Due to the beneficial role of apple phenolics, it is also called as a “new agrochemical crop”. Apple possesses many health beneficial properties for human beings as it is a rich source of phenolic compounds.It has been associated with reducing the risks of certain diseases such as cancers, particularly prostate, liver, colon, and lung cancers, cardiovascular diseases, coronary heart diseases, asthma, type-2 diabetes, thrombotic stroke, and ischemic heart disease. The second chapter of this thesis describes the construction of genetic linkage maps of the parents of a segregating population derived from the cross between the cultivars ‘Prima’ and ‘Fiesta’. For this purpose the already available linkage maps, as described in this chapter, were made denser by inclusion of 240 Diversity Array Technology (DArT) markers. Thus the total number of markers for ‘Prima’ and ‘Fiesta’ integrated map reached to 820. DArT-markers are hybridization based dominant DNA-markers. DArT provides a high-throughput whole genome genotyping platform for the detection and scoring of hundreds of polymorphic loci without any need for prior sequence information. This is the first report on DArT in horticultural trees. Genetic mapping of DArT markers in two mapping populations and their integration with other marker types showed that DArT is a powerful high throughput method for obtaining accurate and reproducible marker data, at low cost per data point. This method appears to be suitable for aligning the genetic maps of different segregating populations. Sequencing of the marker clones showed that they are significantly enriched for low copy, gene rich regions. Chapter 3 describes metabolic diversity of Malus. Wild germplasm was compared to advanced breeding selections and to the segregating F1 population from the cross between the cultivars ‘Prima’ and ‘Fiesta’. The metabolic profiles were analyzed by means of liquid chromatography-mass spectrometry (LC-MS). LC-MS is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. This resulted in the detection of 418 putative metabolites in the peel and 254 in the flesh. Fruits from 23 wild species, eight advanced selections and the segregating F1 population were analyzed. The data were subjected to Principle Components Analysis (PCA). Variance analysis of the first PC showed that genetic variation accounted for 96.6 % in peel and 97.4 % in flesh of the total metabolic variation. Technical variation accounted for 1.4 % and 0.8%, while environmental variation accounted for 2.0% and 1.8% in peel and flesh respectively. The genetic variation between wild genotypes was very large, compared to the advanced selections and the F1 progeny. Only 8 % of the genetic variation of the first principle component was captured by the advanced selections. This indicates strong genetic erosion during breeding. This genetic erosion was mainly caused by reduction of the levels of several flavonoids including catechin, epicatechin and procyanidins. PCA of the F1 progeny of the ‘Prima’ x ‘Fiesta’ cross showed a clear 3:1 Mendelian segregation of metabolites. These metabolites were 4.2 fold less in both peel and flesh in progeny that had inherited the recessive alleles of a gene at the top of Linkage Group16 (LG16) from the heterozygous parents. We found a separate group of 11 metabolites in peel and 12 in flesh. These metabolites were putatively identified as glycosylated forms of b-glycols: R-octane-1, 3-diol and its unsaturated form R-5-(Z)-octene-1, 3-diol which have a potential role in controlling infection by microorganisms and influence the aroma of some ciders. The levels of these metabolites were up to 50 fold more abundant in some progeny compared to both parents. Genetic mapping showed that this strong increase was caused by one locus at the top of LG8, in progeny that had inherited only the recessive alleles of that locus from the heterozygous parents. This research illustrates not only the strong genetic erosion in apple breeding regarding metabolic diversity, and strong reduction of flavonoids in some progeny, but also shows that inbreeding can lead to a strong increase of metabolites that were present at much lower levels in both parents and advanced selections. This loss and gain of metabolites was especially observed in case of accumulation of recessive alleles during inbreeding. The genetic factors controlling metabolite composition were studied in more detail in Chapter 4. We investigated the genetic factors of the quantitative variation of these potentially beneficial compounds (Chapter 3, 4), by combining the genetic maps (Chapter 2) with the LC-MS data for thesegregating F1 population from the cross ‘Prima’ x ‘Fiesta’. This resulted into metabolite quantitative trait loci (mQTLs). When using the software MetaNetwork, 669 significant mQTLs were detected: 488 in the peel and 181 mQTLs in the flesh. Four linkage groups (LGs) i.e. LG1, LG8, LG13 and LG16 were found to contain mQTL hotspots, mainly regulating metabolites that belong to the phenylpropanoid pathway. These include various metabolites i.e. sinapate hexoside, coumaroyl hexoside, phloridzin, quinic acids, phenolic esters, kaempferol glycosides, quercetin glycosides, cyanidin pnetoside, flavan-3-ols (catechin, epicatechin), and procyanidins. The genetics of annotated metabolites was studied in more detail using MapQTL®. It was found that quercetin conjugates had mQTLs on LG1 and LG13. The most important mQTL hotspot with the largest number of metabolites was, however, detected at the top of LG16: mQTLs for 32 peel-related and 17 flesh-related phenolic compounds. The metabolites that mapped in the mQTL hotspot on LG16 all belong to the phenylpropanoid pathway of secondary metabolites. These compounds showed a monogenic Mendelian inheritance in a 3:1 segregation ratio. Procyanidins dimer II was used as a representative of the numerous compounds that mapped at the LG16 mQTL hotspot. By means of graphical genotyping of this monogenic trait, a genetic window could be made in which the gene that caused the mQTL hotspot should reside. We located structural genes involved in the phenolic biosynthetic pathway, using the genetic map together with the published whole genome sequence of apple. The structural gene leucoanthocyanidin reductase (MdLAR1) was detected in the mQTL hotspot window on LG16, as were seven transcription factor genes. To our knowledge, this is the first time that a QTL analysis was performed on such a high number of metabolites in an outbreeding plant species. The expression of the candidate genes found in the mQTL window on LG16 was studied and discussed in Chapter 5. qPCR was used for this purpose and it was found that the expression of only the structural gene MdLAR1 was strongly positively correlated with the metabolite procyanidin dimer II content. Neither the expression profiles of other structural genes of the phenylpropanoid pathway, the transcription factor genes at the mQTL hotspot, nor of transcription factor genes outside the mQTLs hotspot, showed any significant correlation with the procyanidin dimmer II content that mapped at the mQTL hotpot. This indicates that MdLAR1 was the gene, which caused this mQTL hotspot (Chapter 5). The progeny that had inherited one or two copies of the dominant alleles (Mm, MM) showed on the average a 4.4 and 11.8 fold higher expression level of MdLAR1 respectively, compared to the progeny that had inherited the recessive alleles only (mm). This led to a 4.0 fold increase of procyanidin dimer II level at the ripe stage. Strikingly, at the mQTL hotspot at the top of LG16, there is also a locus that controls acidity of the ripe fruits. However, the dominant alleles for acidity appeared to be in repulsion to the dominant alleles for high metabolite levels (Chapter 6). This shows that acidity is controlled by another gene than the metabolite levels. The combination of the genetic position based on the whole apple genome sequence, annotation of potential genes, and expression profiling indicated that the malic acid transporter gene MdALMT2 was responsible for the clear differences in malic acid content and pH in mature apple fruits of the segregating F1 population. The genetic inheritance of at least one dominant allele (MaMa/Mama) of this gene sufficed for a three-fold increase of the malic acid concentration and a reduction of the pH from 4 to 3 in ripe apples, compared to the presence of only the lower expressed recessive allele (mama). This malic acid transporter gene is located at the top of LG16.Malic acid is the predominant organic acid associated with the pH in apple fruits. It is synthesized in the cytoplasm and transported into the cell vacuole. The concentration of malic acid in the cell vacuole determines the pH of the cell. pH is very important for the overall taste of many fruits, including apple, and has profound effects on the organoleptic quality of apples. The pH of mature apples was genetically mapped on LG16 in the segregating population from the cross ‘Prima’ x ‘Fiesta’. To our knowledge, this is the first time that the genetic segregation of the pH in apple is assigned to a specific gene. Further, this gene has not been reported yet in conjunction to pH of apples or other fruits. After cloning of the MdALMT2 gene, it can be used for, proof of principle, influencing the acidic of existing varieties either by silencing this gene in more acidic cultivars or by inserting this gene into the low acidic cultivars. Another step would be to develop an allele specific molecular marker for selection (Marker Assisted Selection) of the acidity of fruits already at seedling stage, five years before the trees carry fruits. In another study, a dominantly mutated allele of the transcription factor gene MdMYB10,including its upstream promoter, coding region and terminator sequence, was introduced by transformation into apple, strawberry and potato plants. The dominantly inherited mutant allele of MdMYB10 from apple induces anthocyanin production throughout the plant, also at the early stage after transformation. The aim was to determine whether MdMYB10 could be used as a visible selectable marker for plant transformation as an alternative to chemically selectable markers, such as kanamycin resistance. After transformation, the color of calli, shoots and well-growing plants were evaluated. Red and green shoots were harvested from apple explants and examined for the presence of the MdMYB10 gene by PCR analysis. Red shoots of apple explants always contained the MdMYB10 gene but not all MdMYB10 containing shoots were red. Strawberry plants transformed with the MdMYB10 gene showed anthocyanin accumulation in leaves and roots. No visible accumulation of anthocyanin could be observed in potato plants grown in vitro, even the ones carrying the MdMYB10 gene. However, acid methanol extracts of potato shoots or roots carrying the MdMYB10 gene contained up to four times higher anthocyanin content than control plants. Therefore, anthocyanin production as a result of the dominant MdMYB1010 gene can be used as a selectable marker for apple, strawberry and potato transformation, replacing kanamycin resistance gene such as nptII. We reported this MdMYB10 as a cisgenic selectable marker gene for apple transformation (Chapter 7). The results from all experimental chapters have been discussed in a broader sense in the general discussion (Chapter 8). The future prospectives and potential challenges in the genetical metabolomics are also highlighted. The approaches we developed in the current thesis could be used not only for developing potentially a more healthy and improved apple but can also be applied for the genetical metabolomics studies in other important crops.

The apple is the most common and culturally important fruit crop of temperate areas. The elucidation of its origin and domestication history is therefore of great interest. The wild Central Asian species Malus sieversii has previously been identified as the main contributor to the genome of the cultivated apple (Malus domestica), on the basis of morphological, molecular, and historical evidence. The possible contribution of other wild species present along the Silk Route running from Asia to Western Europe remains a matter of debate, particularly with respect to the contribution of the European wild apple. We used microsatellite markers and an unprecedented large sampling of five Malus species throughout Eurasia (839 accessions from China to Spain) to show that multiple species have contributed to the genetic makeup of domesticated apples. The wild European crabapple M. sylvestris, in particular, was a major secondary contributor. Bidirectional gene flow between the domesticated apple and the European crabapple resulted in the current M. domestica being genetically more closely related to this species than to its Central Asian progenitor, M. sieversii. We found no evidence of a domestication bottleneck or clonal population structure in apples, despite the use of vegetative propagation by grafting. We show that the evolution of domesticated apples occurred over a long time period and involved more than one wild species. Our results support the view that self-incompatibility, a long lifespan, and cultural practices such as selection from open-pollinated seeds have facilitated introgression from wild relatives and the maintenance of genetic variation during domestication. This combination of processes may account for the diversification of several long-lived perennial crops, yielding domestication patterns different from those observed for annual species., Author Summary The apple, one of the most ubiquitous and culturally important temperate fruit crops, provides us with a unique opportunity to study the process of domestication in trees. The number and identity of the progenitors of the domesticated apple and the erosion of genetic diversity associated with the domestication process remain debated. The Central Asian wild apple has been identified as the main progenitor, but other closely related species along the Silk Route running from Asia to Western Europe may have contributed to the genome of the domesticated crop. Using rapidly evolving genetic markers to make inferences about the recent evolutionary history of the domesticated apple, we found that the European crabapple has made an unexpectedly large contribution to the genome of the domesticated apple. Bidirectional gene flow between the domesticated apple and the European crabapple resulted in the domesticated apple being currently more similar genetically to this secondary genepool than to the ancestral progenitor, the Central Asian wild apple. We found that domesticated apples have evolved over long time scales, with contributions from at least two wild species in different geographic areas, with no significant erosion of genetic diversity. This process of domestication and diversification may be common to other fruit trees and contrasts with the models documented for annual crops.

Effectiviteit bepalen van electrolysewater ter bestrijding van schurft, Venturia inaequalis op appelbladeren.

In een literatuurstudie werd gezocht naar de achtergronden van vruchtzetting en vruchtrui bij appel en peer. Er wordt een overzicht gegeven van vier modellen die zijn ontwikkeld en in de literatuur zijn beschreven om de mate van vruchtrui, zowel de natuurlijke rui als die volgend op een behandeling met chemische dunmiddelen, te voorspellen.

Traditionele veredeling is bij fruitbomen enorm tijdrovend. Veredeling met behulp van genetische modificatie kan het proces erg versnellen, maar dit stuit in Europa op veel maatschappelijke weerstand. Appelveredelaar Henk Schouten van PRI denkt met cisgenese alleen de voordelen van genetische modificatie te introduceren in de traditionele appelveredeling.

Traditionele veredeling is bij fruitbomen enorm tijdrovend. Veredeling met behulp van genetische modificatie kan het proces erg versnellen, maar dit stuit in Europa op veel maatschappelijke weerstand. Appelveredelaar Henk Schouten van PRI denkt met cisgenese alleen de voordelen van genetische modificatie te introduceren in de traditionele appelveredeling.

In een literatuurstudie werd gezocht naar de achtergronden van vruchtzetting en vruchtrui bij appel en peer. Er wordt een overzicht gegeven van vier modellen die zijn ontwikkeld en in de literatuur zijn beschreven om de mate van vruchtrui, zowel de natuurlijke rui als die volgend op een behandeling met chemische dunmiddelen, te voorspellen.

Het Productschap Tuinbouw financiert dit onderzoek

Het Productschap Tuinbouw financiert dit onderzoek

Ondanks de inspanning om vruchtboomkanker onder controle te houden is het verlies van bomen en de productie bij aantasting groot. Bestrijdingsmiddelen kunnen niet meer ingezet worden. Een waarschuwingasmodel (zoals bij de bestrijding van appelschurft) zou de inzet van de resterende bestrijdingsmiddelen moeten kunnen optimaliseren.

Ondanks de inspanning om vruchtboomkanker onder controle te houden is het verlies van bomen en de productie bij aantasting groot. Bestrijdingsmiddelen kunnen niet meer ingezet worden. Een waarschuwingasmodel (zoals bij de bestrijding van appelschurft) zou de inzet van de resterende bestrijdingsmiddelen moeten kunnen optimaliseren.

This project is financed by the Dutch Ministry of Agriculture, nature and Food Quality and is part of the research programme 'Sustainable and competetive agriculture supply chains in pre- and post-European Union accession countries (EU access)'

This project is financed by the Dutch Ministry of Agriculture, nature and Food Quality and is part of the research programme 'Sustainable and competetive agriculture supply chains in pre- and post-European Union accession countries (EU access)'

Stand van zaken bij het onderzoek van PPO Randwijk en PRI naar de werkzaamheid van plantaardige oliën en feromonen voor de bestrijding van de roze appelluis in appel. De werking is gebaseerd op geurstoffen die het paringsgedrag van mannelijke en vrouwelijke bladluizen beïnvloeden

Binnen de biologische fruitteelt is het aantal gewasbeschermingsmiddelen dat toegepast mag worden zeer beperkt. De meest belangrijke ziekte waar tegen bestreden moet worden, is schurft (Venturia inaequalis). Het middel zwavel is het meest toegepaste fungicide dat op dit moment wordt ingezet op appel. In dit rapport wordt een onderzoek beschreven over de regenvastheid van zwavel.

Om het milieu en in het bijzonder het oppervlaktewater zo min mogelijk te belasten met gewasbeschermingsmiddelen, worden door de fruitteeltsector grote inspanningen verricht om de emissie naar het milieu beperkt te houden. Dit rapport beschrijft het verschil in emissie bij gebruik van de Mittererspuit (met of zonder lamellen) met de emissie bij het gebruik van een referentie spuitmachine. Dit is getest in een appelboomgaard.

Om het milieu en in het bijzonder het oppervlaktewater zo min mogelijk te belasten met gewasbeschermingsmiddelen, worden door de fruitteeltsector grote inspanningen verricht om de emissie naar het milieu beperkt te houden. Dit rapport beschrijft het verschil in emissie bij gebruik van de Mittererspuit (met of zonder lamellen) met de emissie bij het gebruik van een referentie spuitmachine. Dit is getest in een appelboomgaard.

The ultimate objective of the production, handling and distribution of fresh fruits and vegetables is to satisfy consumers requirements. In general the attractiveness of fruits and vegetables to consumers is determined both by visible (e.g. colour) and invisible (e.g. healthiness) quality attributes. Flavonoids and hydroxycinnamic acid derivatives, secondary metabolites, contribute largely to both fruit colour and, through fruit consumption, to human health and it is, therefore, very useful to study factors that affect these substances with the aim of further improving the relevant fruit attributes. Flavonoids and hydroxycinnamic acid derivatives are widespread in the plant kingdom, comprise a large group of naturally occurring antioxidants that form part of the human diet. There is considerable evidence for the role of antioxidant constituents of fruits and vegetables in the maintenance of health and disease prevention. Recent studies have shown that the majority of the antioxidant activity of a fruit or vegetable may originate from the flavonoids and other phenolic compounds. Apple fruit are rich in flavonoids such as flavonols (quercetin 3-glycosides), flavanols (catechin, epicatechin, gallocatechin, procyanidins and its polymers), dihydrochalcone glycosides (phloritin glucoside (phloridzin) and phloritin xyloglucoside), and cyanidin 3-glycoside (anthocyanins). Apple fruits also contain considerable amounts of hydroxycinnamic acid derivatives mainly represented by chlorogenic acid. The red colour of apples is primarily a consequence of the flavonoid pigments anthocyanins which are located in the vacuole. Despite the importance of flavonoids for the intrinsic quality of apples very little is known of their regulation in fruit. The aim of the work described in this thesis was therefore, to obtain knowledge on the extent to which the contents of flavonoids and chlorogenic acid in the skin of apples varies, how they develop during fruit growth phase, ripening phase and post harvest phase and how they can be manipulated.Chapter 1 contains a review of the literature. It appears that the accumulation of flavonoids and phenolic acids in plants is under control of many internal and external factors.In Chapter 2 the extent of natural variation in flavonoids and chlorogenic acid concentration due to within fruit, within tree, between orchards, between cultivars and among mutants was determined. Considerable variation was observed among these variables. Individual flavonoids and chlorogenic acid concentrations were not equally distributed within the fruit. Quercetin 3-glycosides and anthocyanin were almost exclusively found in the skin. The sun-exposed skin of individual fruit had much higher cyanidin 3-galactoside (anthocyanin) and quercetin 3-glycoside concentrations than the shaded skin, while phloridzin, catechins and chlorogenic acid were similar in the skin of both sides (Chapter 2). Significant genotypic variation was observed for the concentration of flavonoids and chlorogenic acid. 'Jonagold' apples contain significant higher concentrations (about 30% higher) (Chapters 2 and 6) and amounts (about 2-fold higher) (Chapter 6) of the total flavonoids than 'Elstar' apples. Chlorogenic acid concentration was about 3-fold higher in 'Jonagold' than in 'Elstar' apples. However 'Elstar' apples contained significant higher concentrations of some quercetin glycosides types as quercetin 3-rhamnoglucoside (about 2-fold higher) and quercetin 3-glucosides (about 30% higher) than 'Jonagold' apples. This might be relevant with respect to differences in bio-activity and antioxidant capacity of various flavonoid compounds. As far as the potential maximum concentration of flavonoids in apple is genetically determined breeding would be an important tool for increasing healthiness of apples. The differences between basic forms and coloured mutants within a given cultivar (for example Jonagold-Jonaprince; Elstar-Elshof) show that the potential anthocyanin accumulation (but only that) may increase several fold without influencing the concentrations of other flavonoid classes (Chapter 2). Microscopic study showed that the most blushed mutants had a higher number of red cells per cell layer and more cell layers containing red cells than the standard cultivar and the less blushed mutants. It is striking to observe coloured and completely uncoloured cells as neighbours. Since the selection of coloured mutants is inherently based on the amount of red coloration, selection of mutants for higher levels of other potential healthy flavonoid classes e.g. quercetin 3-glycosides could be considered, providing that such characteristics can be relatively easily determined. The concentrations of anthocyanin, quercetin 3-glycosides and total flavonoids were highest in fruit borne in the top of the tree followed by fruit from the outer tree parts, whereas the lowest concentrations were found in fruit from the inner tree. Terminal fruit contained the highest concentrations of these compounds, including catechins, compared to lateral and spur fruit. Phloridzin and chlorogenic acid were not affected by the position of the fruit in the tree nor by the bearing wood type. The maximum possible difference in flavonoid concentrations, based on difference between top fruit (optimal light conditions) and inner fruit (minimal light conditions) may be 3-fold for quercetin 3-glycosides and 2-fold for total flavonoids (Chapters 2 and 3). There were significant differences in flavonoid and chlorogenic acid concentrations in 'Elstar' fruit between two normally productive orchards differing mainly in growth vigour and internal shading. All these results show that light conditions are a main regulatory factor in the biosynthesis of flavonoids in apples.In Chapter 3 the natural distribution of light within the tree canopy in relation to the concentration of flavonoids and chlorogenic acid in fruit skin was analysed. The concentrations of cyanidin 3-galactoside and quercetin 3-glycosides and the percentage of blush in the fruit skin were directly related to light level in the direct vicinity of the fruit. Light in the interior of the canopy was poorer in UV-A, blue, green and red (R) but richer in far-red (FR) light than at all other positions. Consequently, the FR/R ratio (with large influence on formative processes) was much larger at the interior of the canopy than at all other positions. There was a critical FR/R ratio of about 1 above which no anthocyanin and only low amounts of quercetin 3-glycosides were formed.In Chapter 4 the relationships between the fruit nutrients N, P, K, Ca and Mg and concentrations of flavonoids and chlorogenic acid in fruit skin were studied with two types of 'Elstar'. In an experiment with the mutant 'Elshof' with the 5 nutrients applied at 5 rates in 4 replications, only N and Ca applications resulted in higher concentration of these nutrients in the fruit, but sufficient variation was present among treatments to correlate the concentration of the other nutrients with those of flavonoids and chlorogenic acid. Negative correlations were frequently found between the concentration of N and Mg and the N/Ca ratio in fruit during growth, and anthocyanin and total flavonoids concentration at maturity in 1996, 1997 and 1998. In 1997, these correlations were weakest but still significant. In that season, P and K concentration were frequently negatively correlated with the concentration of anthocyanin and total flavonoids. The concentration of Ca was not related to the concentration of anthocyanin and total flavonoids. In a study in 1996 with standard 'Elstar', we used the variation in nutrient concentration due to differences in fruit position on tree. The concentrations of N and K and the N/Ca ratio in fruit at maturity were negatively and that of Ca was positively correlated with the concentration of anthocyanin and total flavonoids. Magnesium concentration was negatively correlated with anthocyanin concentration but not with total flavonoids. As a consequence of the relation with position of the fruit in the tree an interaction with the influence of light may, however, be expected. Multiple regression models mainly containing N as factor accounted for up to 40% and 30% of the variance in anthocyanin and total flavonoids concentration of 'Elshof' mutant apples, and for up to 70% and 65% of the variance in anthocyanin and total flavonoids concentration of standard 'Elstar' apples. The relationships between plant nutrients and chlorogenic acid concentration in apples were not consistent and further study is required. It is concluded that, in addition to improving light conditions, the concentration of flavonoids in fruit skin could be further increased by optimising fertilization especially that of N, directed at preventing excess N accumulation.In Chapter 5 we tested the concept that under condition of high carbon supply, plants may increase the formation of their secondary metabolites, like phenolic compounds. In field experiments crop load was manipulated by applying flower or fruit thinning at different stages of development and at different severity. At a low crop load, fruit weight, soluble solids, acidity and firmness were significantly higher than at high and moderate loads. However, the concentrations of flavonoid and chlorogenic acid were similar at the different levels of crop load. Time of thinning had no significant influence on the concentration of flavonoids and chlorogenic acid in fruit skin and had no further effect on fruit quality characteristics such as weight, soluble solids, acidity and firmness. Removal of only the interior fruits (about one-third of total fruit) at about 4 weeks before expected commercial harvest had no influence on the concentration of flavonoids and chlorogenic acid or on the quality characteristics of the remaining exterior fruits of either 'Elstar' or 'Jonagold'. The results indicate that, within the 'normal' range of conditions, assimilate availability is not a major regulatory factor in flavonoids and chlorogenic acid formation in apples. These results are in agreement with the lack of any influence of the supply of precursors in the orchard (Chapter 7).In Chapter 6 the changes that take place in the concentration and the amount of individual flavonoids and chlorogenic acid in the skin of 'Elstar' and 'Jonagold' apples during development and ripening were investigated. In both cultivars, the concentration on a dry weight basis of quercetin glycosides, phloridzin and chlorogenic acid was highest early in the season but decreased at different rates during fruit development to reach a steady level during maturation and ripening. Catechins (catechin plus epicatechin) concentration showed a similar pattern, but a temporary increase was observed in an early stage of development. The concentration of cyanidin 3-galactoside (anthocyanin) was relatively high early in the season, gradually decreased to a very low steady level during growth, but started to increase near maturation, especially in the outer fruit. On a fruit basis the amount of quercetin glycosides increased during development and was about two times higher in 'Jonagold' compared to 'Elstar', both in outer and inner fruit. These compounds were the most abundant flavonoids in the skin of both cultivars and their accumulation showed a strong dependency on fruit position on tree. The amount of the second most abundant flavonoid type, catechins, increased during development to a maximum and then showed some decrease by mid season which was independent of fruit position on tree. The amount of phloridzin increased only early in the season reaching a steady level during development and ripening, and was independent of fruit position on tree. The amount of chlorogenic acid in both cultivars initially increased, but subsequently decreased to reach a low steady level and was slightly higher in outer than in inner fruit. The latter phenomenon is the only direct evidence for (net) breakdown of any of the studied phenolics. Although anthocyanin concentration was relatively high at early stages of development, significant accumulation on a fruit basis only occurred during maturation and ripening. The accumulation of anthocyanin, similar to that of quercetin glycosides, showed a strong dependency on fruit position on tree. The results indicate that, in general, the overall production of total flavonoids, with the exception of anthocyanin, and chlorogenic acid in apple skin is completed during fruit development before the onset of maturation.Chapter 7 reports the influence of exogenous application of a number of chemicals that are precursors of flavonoids or are known to affect ripening on the accumulation of flavonoids and chlorogenic acid in 'Jonagold' apple skin with emphasis on anthocyanin. One aim was to identify a possible substrate limitation and another to separate the formation of anthocyanin from other related maturity/ripening events. Since the occurrence of the second peak in anthocyanin formation more or less parallels the maturation and ripening phase (like starch degradation and aroma production), anthocyanin formation itself is often considered as a ripening phenomenon triggered by ethylene. Our results suggest, however, that there is no simple relation to ripening and consequently to ethylene production (though we did not measure ethylene). This is concluded from the promotion of anthocyanin formation by ethephon (an ethylene releasing compound) and the retardation of anthocyanin formation by ABG and GA 3 (known to lower or counteract endogenous ethylene), without significantly altering starch degradation and changes in streif index (combination of starch index, firmness and sugar concentration). Our results have also shown that the other flavonoid classes quercetin 3-glycosides, catechins and phloridzin and chlorogenic acid do not respond to any of the applied chemicals. It is concluded that anthocyanin formation is dependent on developmental signals and independent of both fruit maturity/ripening and of the synthesis of other flavonoid classes and responds in a complicated way to ethylene.In Chapter 8 the changes in individual flavonoids and chlorogenic acid during regular (RS) or ultra low oxygen (ULO) storage conditions at 1°C are reported in both 'Jonagold' and 'Elstar' apples. It could convincingly be shown that during storage of both 'Jonagold' (3, 6 and 8 months) and of 'Elstar' (2, 4 and 6 months) and during 1 or 2 weeks shelf life, the concentrations of cyanidin 3-galactoside and quercetin glycosides were relatively constant, while the concentrations of catechins, phloridzin and chlorogenic acid showed only minor decreases. Moreover there were no significant differences in the concentration of flavonoids and chlorogenic acid between fruits stored under ULO compared to RS conditions. It is concluded that, following harvest, flavonoids present in apples are stable. There is no direct or indirect proof for breakdown (net metabolic turnover) during storage and shelf life.In Chapter 9 the practical applications of the findings made in this study were discussed. Our results show that there is much room for increasing the level of potential health phytochemicals in apples. The first step would be cultivar selection either from already available genotypes or by developing new cultivars through classical breeding or molecular biology and gene technology. We showed that light has a significant impact on the final level of flavonoids in fruit. Therefore, the second and more proximate option would be the optimisation of light conditions within tree canopy by measures such as choice of root stocks, planting system, row orientation and training and pruning systems or covering the orchard floor with reflecting films (though the latter is not promoting the visual aspect of the orchard). A third step could be optimisation of the fertilization programme especially avoiding excess N and better timing of N-application. A further possibility is to sort fruit in healthiness classes. As long as a simple method to detect non-destructively quercetin 3-glycosides is lacking, sorting of fruit based on their blush might be a way to make healthiness classes, since blush is a good marker for exposure to light during growth and thus to some extent for the quercetin 3-glycosides level. Even when cultivar choice and cultivation methods succeed in getting high levels of flavonoids in fruit still the treatment by the consumer determines how much of these substances will be consumed. Many consumers still peel the fruit before consumption thereby removing almost all anthocyanin and quercetin 3-glycosides (Chapter 2). Promotion of fruit on the basis of healthiness is, in our opinion, however, only useful if it is accompanied with a guarantee of absence of pesticides, as is most credible, at least to the public, in organic farming.Because of the large influence of a number of factors at several steps of the production chain, a quantitative model e.g. integrated with light distribution models, would offer a practical and effective tool for estimating the effect of certain measures and to predict and maximise the final level of healthy compounds in apples enabling the development of more accurate intake data and dietary recommendations.

Het Productschap Tuinbouw financiert dit onderzoek

Genetic analysis in strictly outcrossing species is far more complicated than in species that can be selfed. When, in addition to that, a species has a long generation cycle, and when progenies from crosses require much space, as is the case for apple trees, then acquiring genetic knowledge about the species is a matter of high costs and much patience. However, for a breeder such knowledge is highly valuable, since it enables him or her better to combine desired characters into new cultivars.DNA techniques developed in the eighties and nineties have made it possible to visualize variation on the DNA level, e.g. between the two parents of a cross. These differences, usually visualized as differences in banding patterns on a photographic film, are called molecular markers. Molecular markers correspond to physical locations on chromosomes, the marker loci. By studying a progeny of a cross for the absence and presence of combinations of markers, the order of the marker loci on the chromosomes can be estimated and presented in a molecular linkage map. Furthermore, molecular markers also allow the estimation of the location of genes for important traits. Many traits are determined by more than a single gene and in addition to that, suffer from strong influence of environmental factors such as the weather and the soil. The contributions of each of the separate genes, the quantitative trait loci or 'QTLs' are not individually observable, but have to be estimated using statistical techniques called QTL analysis or QTL mapping.In this doctoral thesis, properties of genetic analysis with molecular markers in outbreeding species are studied. A molecular linkage map of apple is presented and map positions of some important genes, such as resistance to apple scab, and fruit acidity, were estimated. Furthermore, quantitative traits in apple were studied and QTLs for fruit quality traits were mapped. Also, frequentist and Bayesian methods for QTL analysis of multiple QTLs were compared, using data of an apple cross.

Genetic analysis in strictly outcrossing species is far more complicated than in species that can be selfed. When, in addition to that, a species has a long generation cycle, and when progenies from crosses require much space, as is the case for apple trees, then acquiring genetic knowledge about the species is a matter of high costs and much patience. However, for a breeder such knowledge is highly valuable, since it enables him or her better to combine desired characters into new cultivars.DNA techniques developed in the eighties and nineties have made it possible to visualize variation on the DNA level, e.g. between the two parents of a cross. These differences, usually visualized as differences in banding patterns on a photographic film, are called molecular markers. Molecular markers correspond to physical locations on chromosomes, the marker loci. By studying a progeny of a cross for the absence and presence of combinations of markers, the order of the marker loci on the chromosomes can be estimated and presented in a molecular linkage map. Furthermore, molecular markers also allow the estimation of the location of genes for important traits. Many traits are determined by more than a single gene and in addition to that, suffer from strong influence of environmental factors such as the weather and the soil. The contributions of each of the separate genes, the quantitative trait loci or 'QTLs' are not individually observable, but have to be estimated using statistical techniques called QTL analysis or QTL mapping.In this doctoral thesis, properties of genetic analysis with molecular markers in outbreeding species are studied. A molecular linkage map of apple is presented and map positions of some important genes, such as resistance to apple scab, and fruit acidity, were estimated. Furthermore, quantitative traits in apple were studied and QTLs for fruit quality traits were mapped. Also, frequentist and Bayesian methods for QTL analysis of multiple QTLs were compared, using data of an apple cross.

Xyloglucans play an important role in connecting cellulose microfibrils in the primary coli wall of plants, and the resulting cellulose-xyloglucan network is thought to determine the strength of these walls. Xyloglucans were isolated from apple fruit and potato tuber cell wall material by alkaline extraction and their primary structures were determined. Major differences between these two polysaccharides were their degree of backbone branching and the presence of fucosyl and arabinosyl residues.The substrate specificity of three ondoglucanases from Trichoderma viride (endol, endolV and endoV) was investigated. The target substrate of endol is cellulose, that of endolV xyloglucan, whereas endoV is the most versatile endoglucanase having the ability of degrading both substrates. EndolV and endoV differ in their mode of action towards potato xyloglucan. Further, strong indications were obtained that xyloglucanase activity is related to a long array of substrate-binding sites.The degradation of the cellulose-xyloglucan network in isolated coli wall material from apple fruit involves several glucanase activities. Xyloglucanase activity is important to make cell wall embedded cellulose more accessible to true cellulolytic enzymes such as endol and cellobiohydrolase. Extensive degradation is required because xyloglucan fragments having a backbone of 20 glucosyl residues (five building units) still bind to cellulose surfaces. These results might explain why fungi excrete so many different kinds of endoglucanases.When the cell walls of living apple fruit tissue were treated with pectin lyase and a mixture of glucanases from Trichoderma viride (liquefaction), the ease with which the apple tissue disintegrated, seemed to depend on the maturity of the fruit. The disintegration of apple fruit tissue during liquefaction correlates to the level of (ripeningrelated) xyloglucan endotransglycosylase activity in apple fruit. An hypothesis for the synergism of fungal and plant glucanases is put forward. Under certain circumstances (controlled liquefaction) the cellulose-xyloglucan network can be modified in such a way, that a stable cellulose-based cloud is formed in the resulting apple juice. The significance of these observations for juice manufacturing is discussed.

For consistent yields in apple fruit production, knowledge of the factors affecting flower bud formation is required. The aim of this study was to gain more insight in the role of endogenous factors in flower bud formation of apple. The effects of temperature, applied gibberellin (GA 4+7 ), the presence of fruits, defoliation and bending on the plastochron (the time interval between formation of successive primordia by the meristern) and the number of appendages per bud at the start of floral differentiation were studied. Although most treatments distinctly affected flower bud formation, the plastochron was only affected by temperature and defoliation. Considerable variation was observed in the number of appendages at the start of floral differentiation, which varied between cultivars, between experiments with the same cultivar and between bud positions. It is concluded that the length of the plastochron and the number of appendages are not critical to the occurrence of flower bud formation.Defoliation inhibited flower bud formation and caused an immediate reduction in the soluble sugar content of the shoot. Bending slightly enhanced flower bud formation, or had no effect, and did not affect the soluble sugar level in the shoot, but increased starch content. The poor correlation between the assimilate level of the shoot and the number of flower buds formed suggests that it is not a main regulatory factor in flower bud formation.It was further investigated whether ammonium affects flower bud formation of apple through affecting endogenous levels of polyamines (PAs). Ammonium and applied PAs have previously been shown to stimulate flower formation of apple. However, due to a lack of effect of ammonium on flower bud formation in the present work, no conclusive evidence could be given. PA levels of buds did not greatly respond to treatments affecting flower bud formation (applied GA 4+7 and shoot bending) despite the fact that both treatments did affect flower bud formation. PA levels did not show distinct changes during the supposed time of floral induction and floral differentiation. It is tentatively suggested that PAs are not major inductive stimuli of flower bud formation of apple.

Xyloglucans play an important role in connecting cellulose microfibrils in the primary coli wall of plants, and the resulting cellulose-xyloglucan network is thought to determine the strength of these walls. Xyloglucans were isolated from apple fruit and potato tuber cell wall material by alkaline extraction and their primary structures were determined. Major differences between these two polysaccharides were their degree of backbone branching and the presence of fucosyl and arabinosyl residues.The substrate specificity of three ondoglucanases from Trichoderma viride (endol, endolV and endoV) was investigated. The target substrate of endol is cellulose, that of endolV xyloglucan, whereas endoV is the most versatile endoglucanase having the ability of degrading both substrates. EndolV and endoV differ in their mode of action towards potato xyloglucan. Further, strong indications were obtained that xyloglucanase activity is related to a long array of substrate-binding sites.The degradation of the cellulose-xyloglucan network in isolated coli wall material from apple fruit involves several glucanase activities. Xyloglucanase activity is important to make cell wall embedded cellulose more accessible to true cellulolytic enzymes such as endol and cellobiohydrolase. Extensive degradation is required because xyloglucan fragments having a backbone of 20 glucosyl residues (five building units) still bind to cellulose surfaces. These results might explain why fungi excrete so many different kinds of endoglucanases.When the cell walls of living apple fruit tissue were treated with pectin lyase and a mixture of glucanases from Trichoderma viride (liquefaction), the ease with which the apple tissue disintegrated, seemed to depend on the maturity of the fruit. The disintegration of apple fruit tissue during liquefaction correlates to the level of (ripeningrelated) xyloglucan endotransglycosylase activity in apple fruit. An hypothesis for the synergism of fungal and plant glucanases is put forward. Under certain circumstances (controlled liquefaction) the cellulose-xyloglucan network can be modified in such a way, that a stable cellulose-based cloud is formed in the resulting apple juice. The significance of these observations for juice manufacturing is discussed.

For consistent yields in apple fruit production, knowledge of the factors affecting flower bud formation is required. The aim of this study was to gain more insight in the role of endogenous factors in flower bud formation of apple. The effects of temperature, applied gibberellin (GA 4+7 ), the presence of fruits, defoliation and bending on the plastochron (the time interval between formation of successive primordia by the meristern) and the number of appendages per bud at the start of floral differentiation were studied. Although most treatments distinctly affected flower bud formation, the plastochron was only affected by temperature and defoliation. Considerable variation was observed in the number of appendages at the start of floral differentiation, which varied between cultivars, between experiments with the same cultivar and between bud positions. It is concluded that the length of the plastochron and the number of appendages are not critical to the occurrence of flower bud formation.Defoliation inhibited flower bud formation and caused an immediate reduction in the soluble sugar content of the shoot. Bending slightly enhanced flower bud formation, or had no effect, and did not affect the soluble sugar level in the shoot, but increased starch content. The poor correlation between the assimilate level of the shoot and the number of flower buds formed suggests that it is not a main regulatory factor in flower bud formation.It was further investigated whether ammonium affects flower bud formation of apple through affecting endogenous levels of polyamines (PAs). Ammonium and applied PAs have previously been shown to stimulate flower formation of apple. However, due to a lack of effect of ammonium on flower bud formation in the present work, no conclusive evidence could be given. PA levels of buds did not greatly respond to treatments affecting flower bud formation (applied GA 4+7 and shoot bending) despite the fact that both treatments did affect flower bud formation. PA levels did not show distinct changes during the supposed time of floral induction and floral differentiation. It is tentatively suggested that PAs are not major inductive stimuli of flower bud formation of apple.

Stand van zaken bij het onderzoek van PPO Randwijk en PRI naar de werkzaamheid van plantaardige oliën en feromonen voor de bestrijding van de roze appelluis in appel. De werking is gebaseerd op geurstoffen die het paringsgedrag van mannelijke en vrouwelijke bladluizen beïnvloeden

The optimization of light interception is essential for the production of highquality fruit. High light interception is required for high yields, whereas insufficient light exposure may lead to inferior flowering and a reduction of many characteristics of fruit quality. Orchard configuration is important for the manipulation of light utilization. In this context, the influence of planting density, arrangement, tree size and shape on light interception was quantified in a model approach. The results were evaluated with the use of datasets from experiments with apple.From the results of the calculation of light absorption and canopy photosynthesis, it could be inferred that early cultivars having a growing season of only four months achieve about the same potential production over a wide range of temperate regions. The production of cultivars with a longer season would, however, increase by at least one tonne fruit (fresh weight) per ha for every degree lower latitude. On the other hand, production at low latitudes may suffer from higher respiration costs.A model for the assessment of light transfer through trees was used to analyse the influence of density, planting system, and tree size and shape. It was concluded that trees with a conical shape have better light penetration into the lower tree parts than have those with parabolic or cylindrical shapes. Light interception and the amount of well-illuminated canopy increase with planting density particularly 9 leaf density of the crowns is not increased. Systems with a low ratio of between-to- within row distance (rectangularity) should intercept light very efficiently.In experiments, planting density was found to be the most important orchard factor for production per ha, which was actually due to leaf area. Systems at low rectangularities produced considerably more well-coloured fruit than did those at higher rectangularities. The product" of three-row bed systems on a triangular design lagged behind that of other systems. A tall bit open tree was found to produce more well-coloured fruit than lower trees with a higher leaf density did. It is concluded that for fruit growers light interception is a key factor for the optimization of orchard management, that can be manipulated by planting density, planting system, and tree size and shape.

Ethyleen is een gasvormig hormoon, dat rijpingsprocessen bij appels in gang zet. Gevolg is dat de vruchten hun hardheid verliezen. Bij Elstar verloopt dat proces snel. Uit onderzoek blijkt dat ethyleen scrubben het verlies aan hardheid reduceert

The optimization of light interception is essential for the production of highquality fruit. High light interception is required for high yields, whereas insufficient light exposure may lead to inferior flowering and a reduction of many characteristics of fruit quality. Orchard configuration is important for the manipulation of light utilization. In this context, the influence of planting density, arrangement, tree size and shape on light interception was quantified in a model approach. The results were evaluated with the use of datasets from experiments with apple.From the results of the calculation of light absorption and canopy photosynthesis, it could be inferred that early cultivars having a growing season of only four months achieve about the same potential production over a wide range of temperate regions. The production of cultivars with a longer season would, however, increase by at least one tonne fruit (fresh weight) per ha for every degree lower latitude. On the other hand, production at low latitudes may suffer from higher respiration costs.A model for the assessment of light transfer through trees was used to analyse the influence of density, planting system, and tree size and shape. It was concluded that trees with a conical shape have better light penetration into the lower tree parts than have those with parabolic or cylindrical shapes. Light interception and the amount of well-illuminated canopy increase with planting density particularly 9 leaf density of the crowns is not increased. Systems with a low ratio of between-to- within row distance (rectangularity) should intercept light very efficiently.In experiments, planting density was found to be the most important orchard factor for production per ha, which was actually due to leaf area. Systems at low rectangularities produced considerably more well-coloured fruit than did those at higher rectangularities. The product" of three-row bed systems on a triangular design lagged behind that of other systems. A tall bit open tree was found to produce more well-coloured fruit than lower trees with a higher leaf density did. It is concluded that for fruit growers light interception is a key factor for the optimization of orchard management, that can be manipulated by planting density, planting system, and tree size and shape.

Ethyleen is een gasvormig hormoon, dat rijpingsprocessen bij appels in gang zet. Gevolg is dat de vruchten hun hardheid verliezen. Bij Elstar verloopt dat proces snel. Uit onderzoek blijkt dat ethyleen scrubben het verlies aan hardheid reduceert