DNA marker-assisted evaluation of fruit firmness at harvest

ORIGINAL PAPERDNA marker-assisted evaluation of fruit firmness at harvest and post-harvest fruit softening in a diverse apple germplasmHilde Nybom &Masoud Ahmadi-Afzadi &Jasna Sehic &Maarten HertogReceived:10May 2012/Revised:12July 2012/Accepted:21July 2012/Published online:11August 2012#Springer-Verlag 2012Abstract Several different genes have been proposed as responsible for fruit texture variability at harvest and/or after storage.We have analysed 127apple cultivars for allelic composition in two key genes that are directly involved in the ethylene biosynthesis pathway,Md-ACS1and Md-ACO1,and two other genes that are involved in cell wall degradation,Md-Exp7and Md-PG1.Firmness was mea-sured with a penetrometer at harvest and after 6or 12weeks (early-and late-maturing cultivars,respectively)of cold storage.Maturation time was positively correlated with firmness at harvest and negatively correlated with fruit softening rate (difference between firmness at harvest and after storage,divided by number of weeks in storage).Polyploid cultivars showed significantly higher firmness at harvest compared to diploids.Alleles previously described as responsible for good texture were associated with signif-icantly lower softening for Md-ACS1and Md-PG1,but the opposite was noted for Md-EXP7.Results were nonsignifi-cant for Md-ACO1.Allele frequencies were very uneven in all four loci,with the three most common multi-locus con-figurations accounting for 64%of the entire material.The predictive power of these genes was calculated with a partial least squares discriminant analysis,and these accounted for 15%of the observed variation in initial firmness and 18%for softening rate.Inclusion of maturation time,storage time (i.e.6or 12weeks)and initial firmness into the model however increased the predictability of softening rate to 38%.Dividing the material in modern (released after 1960)and old cultivars did not change the outcome of our analyses.Keywords DNA marker .Fruit texture .Malus ×domestica .Md-ACO1.Md-ACS1.Md-EXP7.Md-PG1IntroductionFruit texture is a major determinant within the concept of consumer-perceived fruit quality in apple.Various charac-teristics are involved,with high values being desirable for firmness,crispness and juiciness together with very low values for mealiness (Péneau et al.2006).These variables appear to be inter-related since firmer apples are perceived as juicier,crispier,crunchier and less mealy compared to softer fruit (Johnston et al.2002).For very early-maturing cultivars,good texture is needed mainly at harvest since the fruit is usually consumed within a few days.However,for late-maturing cultivars,retaining good texture in cold stor-age becomes increasingly important.Fruit of winter varie-ties is often stored for several months and even up to a year in modern facilities.Moreover,the ability to store well is closely associated with resistance towards fungal storage diseases (Blazek et al.2007)and thus affects grower econ-omy substantially,especially in organic production where chemical fungicides are prohibited.Variables involved in fruit texture are assumed to be under multigenic control (Soglio et al.2009).Analysis of DNA polymorphism in mapping populations has enabled the identification of several quantitative trait loci (QTL)that are associated with fruit firmness at harvest and after storageCommunicated by D.ChagnéH.Nybom (*):M.Ahmadi-Afzadi :J.SehicBalsgård,Department of Plant Breeding and Biotechnology,Swedish University of Agricultural Sciences,Fjälkestadsvägen 459,29194Kristianstad,Sweden e-mail:hilde.nybom@slu.seM.HertogBIOSYST-MeBioS,Katholieke Universiteit Leuven,Willem de Croylaan 42,bus 2428,3001Heverlee,BelgiumTree Genetics &Genomes (2013)9:279–290DOI 10.1007/s11295-012-0554-z(King et al.2000,2001;Liebhard et al.2003;Kenis et al. 2008;McKay2010;Longhi et al.2012).In addition,several candidate genes have recently been identified through infor-mation on ripening physiology and post-harvest changes in fruit texture.Associations between allelic configuration in these genes and parameters like firmness at harvest and/or after storage have been determined in apple genotypes,and co-occurrence with several of the previously identified QTLs has been demonstrated(Costa et al.2010;Longhi et al.2012).Utilisation of markers for these genes in marker-assisted selection(MAS)would be especially valuable in a highly heterozygous,self-incompatible and long-juvenile-period crop like apple.Apple is a climacteric fruit,and loss of firmness during storage,known as fruit softening,is physiologically related to internal levels of ethylene.The two most well-studied fruit firmness genes are Md-ACS1(1-aminocyclopropane-1-carboxylate synthase)and Md-ACO1(1-aminocyclopropane-1-carboxylate oxidase),both of which independently affect the ethylene production of apple cultivars(Oraguzie et al. 2004;Costa et al.2005).Of these two loci,Md-ACS1 appears to have the strongest effect on fruit texture.It has been mapped to LG15in the apple genome(Costa et al. 2005),and the two identified alleles are easily discriminated using PCR and two allele-specific primer pairs(Sunako et al.1999;Harada et al.2000;Oraguzie et al.2004,2007; Nybom et al.2008;Zhu and Barritt2008).Allele2(655bp) is associated with a reduced ethylene production while allele 1(489bp)results in normal ethylene production.The frequency of allele2has increased substantially in apple cultivars introduced during the last century,from below 20%in older cultivars to more than50%in recently registered cultivars,indicating that this allele has been fav-oured by selection for improved fruit quality in modern apple breeding programmes(Nybom et al.2008,2012). Two alleles have been identified also in the Md-ACO1locus, which has been mapped to LG10(Costa et al.2005). Cultivars homozygous for the low ethylene allele1 (525bp)are comparatively firm while both heterozygous cultivars and,especially,cultivars homozygous for allele2 (587bp)show more softening(Costa et al.2005,2010;Zhu and Barritt2008).A direct effect of ethylene on fruit softening is the regu-lation of enzymatic breakdown of cell walls and middle lamellae in the fruit.One of these enzymes is polygalactur-onase which is involved in pectin disassembly,leading to softening of pectin-rich tissue.The ethylene-dependent, biallelic endopolygalacturonase gene Md-PG1has thus been shown to affect fruit softening during ripening in apple (Wakasa et al.2006;Mann et al.2008;Costa et al.2010). This gene has also been mapped to LG10,at a distance of 37cM from Md-ACO1(Costa et al.2010).For detection of the two alleles,a previously undescribed SCAR marker (Broothaerts et al.unpublished)produces one band(350bp) in cultivars that are homozygous for the regular-softening allele1while two bands(120and220bp)are produced in cultivars that are homozygous for the low softening allele2.Expansin enzymes play an important role in the depoly-merisation of different polysaccharides in the cell walls during fruit softening.An expansin homolog,Md-Exp7,has been mapped on LG1,and a functional marker was developed by Costa et al.(2008)based on an SSR motif within this gene. Three alleles were reported:198bp(best firmness retention), 202bp(intermediate)and214bp(worst)(Costa et al.2008). However,a survey of allele frequencies in a large apple collection showed that the202-bp allele has increased from just below60to85%during the last century,suggesting that recent apple breeding has actually favoured this allele rather than the198-bp allele(Nybom et al.2012).Most of the research on these four genes has been carried out in progenies derived from crossing cultivars with con-trasting allelic configurations(see,e.g.Longhi et al.2012). Large-scale screenings of cultivar collections have been undertaken mainly with Md-ACS1and results have been somewhat contradictory,showing associations with either firmness at harvest(Zhu and Barritt2008)or with fruit softening during storage(Costa et al.2005;Oraguzie et al. 2007)or with both variables(Oraguzie et al.2004).The aim of our present study is to evaluate the extent to which variation in fruit softening in a large germplasm collec-tion focused on cultivars for a relatively cool climate can be explained by factors like maturation time and initial firmness, and the allelic configuration in these four major genes. Materials and methodsPlant materialA total of127apple cultivars were sampled,45of which have probably originated in Sweden while the remainder are from all over the world(Table1).Fifty-two cultivars were released after1960(‘modern cultivars’)whereas the rest have been in cultivation before this time(‘old cultivars’). Most of the sampled trees were grown in the fruit tree collections at Balsgard,SLU in southern Sweden but22 samples were obtained from a former field station in Kivik,about60km SW of Balsgard(Table1).In2010, apples were harvested at a pre-climacteric stage to mirror conditions in a commercial orchard where fruit is picked for storage.Harvesting time was determined by an iodine starch test,aiming for starch conversion values of4–5(on a10-point scale)since such fruits are suitable for long-term storage.The first cultivars were harvested on August10, and the last on October26.Maturation time was calculatedTable1The127analysed apple cultivars and year of release(O0 before1960,N0after1960),allelic configuration at four loci,fruit firmness at harvest and after storage,softening rate(difference between firmness at harvest and after storage,divided by number of weeks in storage)and maturation time(day of harvest with day10August10)Cultivars Age Allelic configuration Firmnessat harvest Firmnessafter storageSofteningrateMaturationtimeold/new Md-ACS1Md-ACO1Md-EXP7Md-PG1Stored6weeksBeauty of Bath O1/22202:20218.5 5.40.521 Close3×O12202:20217.6 3.70.651 Gyllenkroks Astrakan O12198:20217.4 4.40.501 Valldaäpple O12198:20218.2 4.90.551 Vista Bella N12202:20217.5 5.20.381 Astrakan red O1/22202:20217.2 5.00.372 Quinte N12202:21017.5 4.50.502 Julyred N12198:2021 6.6 4.30.382 Nanna N11/2198:20217.0 4.80.372 Mantet O1/22202:20218.1 4.40.622 Sylvia N12198:2021 6.9 5.20.282 Agra N12202:21018.1 4.20.654 Transparente Blanche O12198:20218.2 4.80.574 Siv N1/21/2198:2021 5.4 4.50.157 Sävstaholm O12198:20219.4 5.60.637 VittsjöO12198:20217.2 5.50.287 Snövit O1/22202:20217.5 5.60.327 Stäringe Karin O12198:19818.2 4.60.6010 Alice N12198:20218.9 4.70.7014 Discovery N22202:20219.27.00.3714 Josefiner O12202:20218.2 4.20.6714 Katinka N12202:20218.4 5.70.457 Idunn N12202:20217.5 4.90.437 Sörmlands O12202:202110.17.70.407 Domöfavorit O12202:202110.810.30.0810 Aspa O12202:21017.2 5.90.2214 Trogsta O1/22198:20217.7 5.50.3714 Charlamovsky O12202:21018.2 6.20.3314 Samo N12202:2161 6.0 3.90.3514 Antonovka Pamtorutka O12198:20219.410.3015 Eir N1/21/2198:2021 6.8 4.40.4015 Jaspi N12202:20217.4 4.50.4816 William’s Pride N1/22202:20218.6 6.60.3316 Retina N12202:202110.17.40.4516 Katja N1/22202:20218.1 4.80.5521 Witos N1/22202:20218.5 5.60.4821 Lobo O12202:2021/27.9 5.70.3721 Summerred N1/22202:20217.5 4.90.4321 Gravensteiner yellow3×O12198:202:20617.8 6.50.2221 Gravensteiner red3×O12198:202:20617.6 5.80.3021 Veseäpple O12198:20217.4 6.00.2322 Signe Tillisch O1/22202:20219.87.40.4022 Mio O12198:20217.2 3.80.5722 Risäter O12202:22017.8 6.40.2322at harvest after storage rate time old/new Md-ACS1Md-ACO1Md-EXP7Md-PG1Reglindis N1/22202:20219.07.40.2728 Drakenberg O12202:202110.08.10.3228 Redfree N12202:20217.9 6.20.2828 Prima N1/22202:20219.57.00.4228 Guldborg O12198:20817.4 5.30.3528 Landskrona O1/22198:20218.8 5.70.5228 Spässerud O12202:21017.4 5.80.2728 James Grieve O12202:20217.9 4.60.5528 Heta N12202:2161/2 5.0 4.40.1029 Antonovka Kamenitschka O12198:2021/29.711.0029 Birgit Bonnier N12202:2021 6.7 5.30.2329 Sawa N1/22202:2021 6.7 4.50.3731 Linda O1/22202:20217.8 6.20.2731 Rödluvan N1/22198:2021 6.9 4.20.4531 Stored12weeksK:1160N1/22202:2021 6.7 1.80.4135 Honeycrisp N1/22202:21019.0 4.80.3535 Kavlås O12198:2021 6.1 1.80.3635 Eva-Lotta N12202:20217.4 2.50.4135 Fredrik N1/22202:20217.5 2.60.4135 Worcester pearmain O1/22202:20218.3 2.50.4835 Ringstad O1/22198:1981 4.8 2.00.2335 Flädie O12198:20217.2 2.30.4135 FulleröO12202:202111.7 3.60.6835 Filippa O12202:20218.4 2.60.4835 Borgherre O12198:202110.3 3.20.5936 Göteborgs flickäpple O12198:20218.4n.d.-36 McIntosh O12202:20218.3 2.40.4936 Dayton N12202:20218.0 2.80.4337 Ella N1/22202:20218.9 2.40.5437 Aroma N1/22202:20217.6 2.70.4137 Amorosa N1/22202:2021 6.9 2.20.3937 Värmlands sötäpple O12198:20617.6 2.70.4138 Stenkyrke O12202:208110.9 3.00.6638 Gråylling,Skokloster O12198:20218.7 2.60.5138 Zhigulevskoe N12202:21019.9 3.30.5538ÅkeröO12198:19819.0 2.90.5138 Rebella N21/2202:2021/28.0 2.60.4538 Kalmare glas3×O12198:202111.5 6.00.4639 Frösåker3×O12198:202110.2 6.10.3439 Hannaäpple O12198:20218.8 5.50.2839 Prinsessäpple O11/2198:202111.7 3.90.6543 Cox's Orange Pippin O1/22202:20219.1 5.40.3143 Jonagold3×N1/22202:20217.6 3.90.3143 Jonathan O1/21/2202:20219.9 5.80.3443 Blenheim Orange3×O12198:20219.2 5.40.3243 Brunnsäpple O11/2198:2021 6.8 3.80.2543by denoting August10as‘1’and then adding number of days to harvest for each cultivar.Leaves for DNA analyses were,in most cases,collected from the same trees as the harvested fruit.Penetrometer readingsA puncture test,using a penetrometer(model FT-327, Effigy,Alfonsine,Italy,plunger diameter11.1mm,depth 7.9mm)was applied to assess fruit firmness(two measure-ments on opposite sides without skin for10fruits,expressed as kilograms per square centimetre)at harvest,as well as after6weeks of cold storage(+2.5°C,normal atmosphere) for58early-maturing cultivars(i.e.cultivars harvested from August10to September9)and after12weeks of cold storage for69late-maturing cultivars(harvested from September13to October26).The stored fruit was placed in room temperature about4h prior to measurements.Theat harvest after storage rate timeold/new Md-ACS1Md-ACO1Md-EXP7Md-PG1Pinova N22202:2021/28.4 6.20.1843 Boiken O1/22198:202111.8 6.60.4343 Laxton's Superb O12202:20218.4 5.80.2243 Angold N1/22202:2021 6.5 4.00.2143 Holsteiner Cox3×O1/21/2202:20219.5 6.00.2943 Gelber Richard O1/22202:20217.4 5.20.1843 Ribston3×O1/22202:202110.8 6.20.3843 Queen Cox O1/22202:20219.1 5.30.3243 Mutsu3×O1/22198:2021/28.9 5.00.3243 Cortland O12202:20217.1 4.90.1843 Alfa4×O12198:202110.4 6.80.3043 Santana N12202:202110.0 6.20.3243 Richelieu N1/22202:20217.4 4.10.2843 Rubinola N22202:2021/28.0 5.30.2243 Delorina N1/22198:19819.6 6.70.2443 Vrams järnäpple3×O1/22202:202111.4 6.90.3843 Himmelstalund O12198:2021 6.7 3.40.2845 Elise N22202:2021/29.7 6.00.3145 Delicious Classic Red O1/21202:202110.07.00.2549 Dronning Louise O12198:202110.1 4.80.4449 Golden Delicious O1/22202:2021/29.6 5.90.3149 Borsdorfer O12198:202111.67.40.3549 Pigeon O12202:210110.7 5.40.4449 Cox's Pomona O1/22202:2021 6.1 4.00.1849 Boskoop3×O12198:202111.0 6.70.3649 Ingrid Marie O1/22202:20217.9 4.60.2849 Rajka N22202:2021/27.7 4.50.2752 Reanda N1/21/2202:20217.0 5.20.1552 Ananas Reinette O12198:20219.6 6.50.2658 Oretorp O12198:202111.4 6.70.3958 Goldrush N22202:2021/211.79.20.2158 Frida N12202:20218.4 5.80.2257 Sultanat N12202:20219.1 4.80.3659 Zarya Alatau N12202:20219.6 5.40.3559 Karin Schneider O1/22202:20217.8 4.60.2759 Kim N1/22202:20217.1 5.20.1664 Gloster N21/2198:202110.4 6.60.3276decision to use different durations of storage was based on the fact that the texture of early-maturing cultivars would have deteriorated and become difficult to measure if stored longer than6weeks,whereas for late cultivars,some might not have reached the climacteric stage after only6weeks making it difficult to observe differences in firmness loss. The chosen date for splitting the two groups reflects the division between‘summer varieties’which are sold within a few weeks after harvest,and‘fall and winter varieties’which are stored for a couple of months or longer.DNA analysesMost of the investigated cultivars had already been screened for the Md-ACS1locus(Nybom et al.2008,2012)and for the Md-EXP7locus(Nybom et al.2012)using the protocols described in Nybom et al.(2008)and in Costa et al.(2008), respectively.When required,additional analyses for these two genes were conducted as in our previous publications.In addition,the whole material was screened for the Md-PG1locus using a CAPS marker which detected two differ-ent alleles.The PCR protocol(Broothaerts et al.unpub-lished)used a total volume of25μl,containing25ng of DNA,1.5mM MgCl2,0.2mM of each dNTP,0.4μM of both primer forward and reverse,1×PCR buffer and1U Taq DNA polymerase(Thermo Fisher Scientific,Surrey, UK).PCR was performed with VWR UNOCYCLER (VWR,Stockholm,Sweden)programmed for2min at 94°C,35cycles of30s at94°C,30s at58°C and30s at72°C,and a final elongation step of7min at72°C.After PCR,10μl of PCR products was incubated with0.5μl of restriction enzyme Bse DI and2μl buffer Tango™(Fermentas,Thermo Fischer Scientific,St.Leon-Rot, Germany)in a total volume of30.5μl,at55°C for2h. After incubation,the reaction was stopped by cold inactiva-tion(−18°C).The amplified and cleaved products were stained with GelRed™(Biotium Inc.,Hayward,CA),sepa-rated by electrophoresis in a1.8%agarose gel and photo-graphed under UV illumination.Three types of fragments were scored:the uncut fragment of350bp(allele1)and 120bp+220bp(allele2).The whole material was also analysed for Md-ACO1with a SCAR marker,which detected two different alleles(Costa et al.2005).The PCR protocol was changed slightly from the originally described protocol;we used a total volume of 25μl,containing25ng of DNA,0.75mM MgCl2,0.25mM of each dNTP,0.2μM of both primer forward and reverse, 1X PCR buffer and1U Taq DNA polymerase(GE Healthcare,illustra™,Buckinghamshire,UK).PCR was performed with VWR UNOCYCLER programmed for 2min at94°C,35cycles of45s at66°C,2min at72°C and45s at94°C followed by45s at66°C and a final elongation step of10min at72°C.The amplified products were stained with GelRed™,separated by electrophoresis in a1.8%agarose gel and photographed under UV illumina-tion.Two types of fragments were scored:525bp(allele1) and587bp(allele2).Statistical evaluationsFor each cultivar,mean firmness at harvest,mean softening (difference between firmness at harvest and firmness after storage)and mean softening per week of storage(value for mean softening divided by6or12,depending on the storage period)was calculated.Mann–Whitney U tests were used to compare groups of cultivars(with N>6).Dependence of fruit firmness and fruit softening,respectively,on matura-tion time was investigated with regression analysis.Partial least squares discriminant analysis(PLS-DA)was applied to investigate the ability of the multi-locus genotype (the independent variables)to predict the response variables, i.e.firmness at harvest and softening per week of storage.In this method,a multiple linear regression model is fitted by projecting the independent variables and the dependent re-sponse variable to a new variable space as defined by the latent variables,maximising the covariance between the dependent and independent variables.This model defines the multidimensional direction in the genotype space that explains the maximum variance in the response vari-able.PLS-DA was applied using The Unscrambler®X.1 (CAMO Software,2011).ResultsFruit firmness and fruit softening dataWhen data for all127cultivars were analysed together,fruit firmness at harvest(calculated as the mean value of two measurements on each of10fruits)was normally distributed. For fruit softening,calculated as the difference between firm-ness at harvest and firmness after storage,the material was divided into groups:early harvested cultivars(stored for 6weeks)and late harvested(stored for12weeks).For these two groups,the mean firmness at harvest was7.94and 9.01kg/cm2,respectively,while the mean firmness after storage was5.65and4.58kg/cm2,respectively.To enable the comparison of all cultivars,another param-eter fruit softening rate was calculated as fruit softening divided by the number of weeks in storage:0.38for early harvested cultivars and0.37for late harvested.This latter parameter was also normally distributed except for a few of the early harvested cultivars with negligible loss in firmness during storage(Fig.1).Thus‘Antonovka Kamenitschka’and‘Antonovka Pamtorutka’,with relatively high initial firmness(9.7and9.4kg/cm2,respectively),retained or evenimproved on this firmness according to penetrometer values:11.0and 10.3kg/cm 2,respectively,after 6weeks of storage.In the single-locus data analyses,softening was recorded as ‘0’for these two cultivars.However,they showed strong signs of senescence in the form of a rubbery fruit flesh texture and would probably have been given lower values with a different technical equipment.In the late-maturing group,fruit of ‘Göteborgs Flickäpple ’had deteriorated after storage so that a penetrometer value could not be obtained,and it was therefore excluded from analyses.The two groups of cultivars (‘old ’versus ‘modern ’)were not significantly different in fruit softening as illustrated by Fig.1.Influence of ploidy levelMean values for the 113presumably diploid and 14poly-ploid (Garkava-Gustavsson et al.2008;13triploids and 1tetraploid,Table 1)cultivars were calculated for firmness at harvest and for softening rate (i.e.softening per week of storage)(Table 2).While the difference in firmness at har-vest was very small among the early-maturing cultivars (only three of these were,however,polyploid),a Mann –Whitney U test showed that among late-maturing cultivars,polyploids had significantly firmer fruit at harvest (10.07kg/cm 2)than the diploids (8.81):U 1/U 20150/488,N 1/N 2011/58,df 067,P <0.01.This difference remained also after storage (5.65and 4.38,respectively);U 1/U 20165.5/461.5,N 1/N 2011/57,df 066,P <0.05.By contrast,softening rate was almost identical between diploids (0.38and 0.36for early-and late-maturing cultivars,respectively)and polyploids (0.37for both early-and late-maturing cultivars).Associations between maturation and fruit firmness and softeningFruit firmness at harvest increased significantly with matu-ration time when all 127cultivars were analysed together:y 00.029x +7.559,R 200.108,P <0.001.However,when the material was divided into early-and late-maturing cultivars,respectively,there was no tendency at all among the early-maturing cultivars,and a nonsignificant (but close to the threshold of P 00.05)increase for the late-maturing culti-vars:y 00.043x +7.005,R 200.046,P 00.076.For fruit soft-ening rate,a negative relationship with maturation time was detected when all cultivars were analysed together:y 0−0.003x +0.450,R 200.099,P <0.001,as well as for both the early-maturing cultivars:y 0−0.005x +0.470,R 200.112,P 00.010,and the late-maturing:y 0−0.007x +0.644,R 200.201,P <0.001.Associations between allelic configurations and fruit firmness and softeningAmong the 127cultivars investigated,only eight were ho-mozygous for Md-ACS1allele 2which is associated with low ethylene production (Tables 1and 2).Another 46culti-vars were heterozygous while 73were homozygous for the regular ethylene production allele,i.e.allele 1.In the early-maturing cultivars,no associations were found between allelic configuration and firmness at harvest (mean values 7.94,7.91and 9.20kg/cm 2),after storage (5.70,5.51and 7.00)or softening rate (0.37,0.40and 0.37).Among the late-maturing cultivars,those homozygous for allele 1had significantly firmer fruits (9.24)at harvest than the hetero-zygotes (7.04):U 1/U 20332/476,N 1/N 2028/34,df 060,P <0.05.As expected,softening rate was,however,higher in cultivars homozygous for allele 1(0.41)when compared to heterozygous (0.20):U 1/U 20279/645,N 1/N 2028/33,df 059,P <0.01,or to those homozygous for allele 2(0.28):U 1/U 2049.5/181.5,N 1/N 207/33,df 038,P <0.05.For Md-ACO1,the majority of cultivars were homozy-gous for the undesirable allele 2while only a single cultivar (‘Red Delicious ’)was homozygous for the low ethylene allele 1(Tables 1and 2).No significant variation among either early-or late-maturing cultivars with the different allele configurations was found,either in firmness at harvest (–,6.40and 8.04kg/cm 2for early-maturing,and 10.0,9.04and 8.84for late-maturing),after storage (–,4.57and 5.72for early-maturing,and 7.00,4.84and 4.58forlate-Fig.1Histograms showing the distribution of fruit softening rate (difference between firmness at harvest and after storage,divided by number of weeks in storage)as observed per storage period and per cultivar group (old versus modern cultivars,i.e.released before or after 1960).The values indicated refer to the average softening rate per group with the intervals indicating the 95%confidence intervals for these averages.The overall average softening rate equals 0.37±0.027kg cm −2week −1maturing)or in softening rate(–,0.30and0.38for early-maturing,and0.25,0.35and0.36for late-maturing).For Md-Exp7,two alleles were very common,198and 202bp,while five additional alleles occurred at lower frequencies(Table1).The most common genotype was 202:202found in68cultivars,provided that these cultivars are homozygous for the202allele and do not carry an undetected allele(Table2).Also quite common was 198:202found in39cultivars,whereas only four cultivars appeared to be homozygous for the198allele.In addition,a total of16cultivars carried at least one of the uncommon alleles.Thus,allele210was found in eight cultivars,allele 206in three cultivars,alleles208and216in two cultivars each and allele220in a single cultivar.No significant differences were found among early-maturing cultivars in firmness at harvest(8.20,7.73,8.36,7.68and6.93kg/cm2), after storage(4.60,5.53,5.90,5.32and5.38)or in softening rate(0.60,0.37,0.41,0.39and0.26).Among late-maturing cultivars,the202:202genotype showed significantly lessfirmness at harvest(8.50)compared to the198:202geno-type(9.56):U1/U20256/584,N1/N2021/40,df059,P< 0.01.However,softening rate was also significantly lowerin the202:202cultivars(0.32):U1/U20285.5/514.5,N1/ N2020/40,df058,P<0.05compared to the198:202(0.38).For Md-PG1,allele2has been described as the mostdesirable.No cultivars were,however,homozygous for thisallele,and only11were heterozygous.Significant differ-ences between homozygous(1:1)and heterozygous(1:2)cultivars were not found in initial firmness(7.85and7.53kg/cm2for early-maturing cultivars and8.86and9.00for late-maturing cultivars)or in firmness after storage(5.59and7.03for early-maturing cultivars and4.52and5.59forlate-maturing cultivars).Mainly due to the unequal samplesizes(N055and N03,respectively),early-maturing culti-vars did not differ significantly in softening rate either(0.38and0.16).For the late-maturing cultivars,a significantlyTable2Number of analysed cultivars,stored at6or12weeks,their genotypes and mean values for fruit firmness at harvest and softening rate (difference between firmness at harvest and after storage,divided by number of weeks in storage)Grouping No.of cultivars Firmness at harvest Firmness after storage Softening rateEarly harvest LateharvestTotal EarlyharvestLateharvestEarlyharvestLateharvestEarlyharvestLateharvestTotal58691277.949.01 5.65 4.580.380.37 All putative diploids5558113(89%)7.978.81 5.67 4.380.380.37 All polyploids31114(11%)7.6710.07 5.33 5.650.390.37 ACS101:1393473(57%)7.949.24 5.70 4.320.370.41 ACS101:2182846(36%)7.917.045,51 4.590.400.20 ACS102:2178(6%)9.209.137.00 5.770.370.28 ACO101:1011(1%)–10.00–7.00–0.25 ACO101:23710(8%) 6.409.04 4.57 4.840.300.35 ACO102:25561116(91%)8.048.84 5.72 4.580.380.36 EXP70198:198134(3%)8.207.80 4.60 3.870.600.33 EXP70198:202182139(31%)7.739.56 5.53 5.060.370.38 EXP70202:202284068(54%)8.368.50 5.90 4.600.410.32 EXP70202:210538(6%)7.689.87 5.32 4.500.390.45 EXP7,remainder628(6%) 6.939.25 5.38 2.850.260.53 PGI01:15561116(91%)7.858.86 5.59 4.520.380.36 PGI01:23811(8%)7.539.007.03 5.590.160.28 ACS1,ACO1,EXP7,PGI1:2,2:2,202:202,1:1131831(24%)8.218.01 5.75 3.830.410.35 1:1,2:2,198:202,1:1111627(21%)7.899.45 5.41 5.010.410.37 1:1,2:2,202:202,1:1131124(19%)9.088.76 5.98 4.250.520.38 1:1,2:2,202:210,1:1527(6%)7.6810.30 5.32 4.350.390.50 2:2,2:2,202:202,1:2055(4%)–9.10– 6.24–0.24 1:2,2:2,198:202,1:1314(3%)7.8011.8 5.13 6.600.440.43 All four loci,remainder131629(23%)7.378.92 5.72 4.620.300.36For groups with<5cultivars,parameter values are given in italics.Parameter values in bold text indicate that Mann–Whitney U test detected a significant difference between groups of cultivars with different genotypes(described in more detail in the text)。