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Cold Sweetening in Diploid Potato: Mapping Quantitative Trait Loci and Candidate Genes

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Cold Sweetening in Diploid Potato: Mapping Quantitative Trait Loci and Candidate Genes
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  Copyright   ©  2002 by the Genetics Society of America Cold Sweetening in Diploid Potato: Mapping Quantitative Trait Lociand Candidate Genes Cristina M. Mene´ndez,* ,1 Enrique Ritter, † Ralf Scha ¨fer-Pregl,* Birgit Walkemeier,* Alexandra Kalde,* Francesco Salamini* and Christiane Gebhardt* ,2 * Max-Planck-Institut fu ¨ r Zu ¨ chtungsforschung, 50829 Ko ¨ ln, Germany and   † NEIKER, Granja Modelo de Arkaute, 01080 Vitoria, Spain  Manuscript received May 1, 2002 Accepted for publication August 9, 2002 ABSTRACT A candidate gene approach has been used as a first step to identify the molecular basis of quantitativetrait variation in potato. Sugar content of tubers upon cold storage was the model trait chosen becausethe metabolic pathways involved in starch and sugar metabolism are well known and many of the geneshave been cloned. Tubersof two F 1 populations of diploid potato grownin six environments were evaluatedfor sugar content after cold storage. The populations were genotyped with RFLP, AFLP, and candidategene markers. QTL analysis revealed that QTL for glucose, fructose, and sucrose content were locatedon all potato chromosomes. Most QTL for glucose content mapped to the same positions as QTL forfructose content. QTL explaining   10% of the variability for reducing sugars were located on linkagegroups I,III, VII,VIII, IX,and XI.QTL consistentacross populations and/orenvironments wereidentified.QTL were linked to genes encoding invertase,sucrose synthase 3, sucrose phosphate synthase, ADP-glucosepyrophosphorylase, sucrose transporter 1, and a putative sucrose sensor. The results suggest that allelic variants of enzymes operating in carbohydrate metabolic pathways contribute to the genetic variation incold sweetening. T HE accumulation of free sugars in plants exposed aldehyde groups of reducing sugars and free   -aminoto low temperatures is a widespread phenomenon groups of amino acids and proteins (T alburt  et al  .that has long been recognized (M u¨ller -T hurgau 1882). 1975), which results in a dark and bitter product. TheThe type of sugars accumulated is species dependent  correlation between the reducing sugar content of tu-(G uy et al  . 1992). Storage organs, such as potato tubers, bers and the extent of browning during processing hasaccumulatebothreducingsugars(glucoseandfructose) been documented (C offin  et al  . 1987; S cheffler  et al  .and sucrose when subjected to chilling temperatures, a 1992).phenomenon known as cold sweetening (B urton 1969). The sugar content of potato tubers is a quantitativeDormantpotatotubersaremetabolicallyinactiveexcept  trait with heritability values ranging from very highfor slow starch degradation and synthesis of sucrose (0.91; G rassert et al  . 1984) to intermediate (0.47–0.63; with energy provided by glycolysis and respiration. Cold P ereira  et al  . 1994). Quantitative traits can be geneti-sweeteningisexplainedasashiftinthebalancebetween cally dissected using linkage maps that are based onstarch degradation and glycolysis, leading to the accu- molecular markers (reviewed by T anksley  1993; S tuber mulation of sucrose (I sherwood  1973), which is then 1995). Quantitative trait locus (QTL) analysis in plantsconvertedintoglucoseandfructose.Althoughthemeta-is performed in segregating populations generated by bolic pathways and enzymes involved in starch-hexoseexperimental crosses. Because the cultivated potato isinterconversion are well characterized, little is knowna tetraploid displaying tetrasomic inheritance, diploidabout their individual contribution to cold sweeteningpotatohasbeenusedtogenerateseveralmolecularlink-and  in vivo   regulation (G reiner  et al  . 1999).age maps for the species (B onierbale  et al  . 1988; G eb- Reducing sugar content in cold-stored potatoes is a hardt  et al  . 1989, 2001; J acobs  et al  . 1995; V  an  E ck  et  major problem for the potato processing industry since al  . 1995; M ilbourne et al  . 1998). These molecular mapsthe industry favors storing tubers at temperatures  10  have been instrumental for locating a number of QTLto delay sprouting. The high frying temperature usedfor tuber-associated traits (F reyre  et al  . 1994; V  an  E ck for the production of potato chips and french fries et al  . 1994; V  an  D en  B erg  et al  . 1996; S cha¨fer -P regl  et  causes a nonenzymatic Maillard reaction between free al  .1998).OneQTLstudyaddressedthecold-sweeteningphenomenon by measuring chip color (D ouches  andF reyre  1994). 1 Present address:   University of La Rioja, 26006 Logron˜o, Spain. Despite the importance of quantitative genetic varia- 2 Corresponding author:   Max-Planck-Institut fu¨r Zu¨chtungsforschung, tion in many areas of plant biology, there is little under- Carl-von-Linne´-Weg 10, 50829 Ko¨ln, Germany.E-mail: gebhardt@mpiz-koeln.mpg.de  standing of the molecular basis that controls this varia- Genetics  162:  1423–1434 (November 2002)  1424 C. M. Mene´ndez  et al. duction, such as those encoding invertases, sucrose phos-phate synthase, and sucrose synthases.The objective of this work was to identify QTL forcold sweetening in the potato genome and to evaluatespecific metabolic genes as candidate genes, as a first step toward identifying genes controlling this economi-cally important trait. MATERIALS AND METHODS Plant material:  Two diploid F 1  populations, H94A andH94C, were used. Population H94A resulted from crossing Figure 1.—Schematicdiagramforsugar/starchmetabolism  Solanumtuberosum  lineH81.839/1,selectedforitslowreducingin potato tubers (modified after S owokinos  2001). Enzymes sugar content (RSC; P  A   P54, seed parent; RSC  0.09%  included are ADP-glucose pyrophosphorylase ( AGP  ), starch 0.01), with line H80.696/4 (P B  P40, pollen parent; RSC  synthase ( SS  ), starch branching enzyme ( Sbe  ), starch phos- 0.26%    0.05). The H94A mapping population consisted of phorylase( STP  ),amylase( AMY  ),glyceraldehyde3-phosphate 146 F 1  genotypes. Population H94C was derived from crossingdehydrogenase ( GapC  ), sucrose phosphate synthase ( SPS  ), the  S. tuberosum   line H82.337/49 (P  A     P18, seed parent;sucrose 6-P phosphatase ( S6P  ), UDP-glucose pyrophosphory- RSC  0.51%  0.19) with line H82.2032/1 (P B  P50, pollenlase( UGP  ),sucrosesynthase( SUS  ),invertase( INV  ),andpyro- parent; RSC  1.83%  0.59). Both parents were unselectedphosphatase ( Ppa  ). Circles indicate the enzymes for which for reducing sugar content. A total of 189 F 1  hybrids wereencoding genes have been mapped in H94A or H94C. For genotyped in this population. The parental lines of both map-map positions of other genes involved in carbohydrate metab- ping populations were highly heterozygous (G ebhardt  et al. olism and transport see C hen  et al  . (2001). 1989). The first tuber generation was obtained from seedlingplants grown in pots in the greenhouse. Tubers were multipliedin pots under Saran cover, to obtain virus-free seed tubers forfield trials. tion (M itchell -O lds  and P edersen  1998). QTL maps Field trials and experimental design:  In 1996, populations are the first step toward the identification of genes re-  H94A and H94C were grown at two locations in Germany: sponsible for QTL, either by positional cloning (F rid-  Carolinensiel, an experimental field close to the North Seacoast, and Scharnhorst, the Max-Planck-Institute’s field sta- man etal. 2000;F rary etal. 2000)orbyacandidategene tion. At Carolinensiel, 10 tubers per clone were planted in a approach. The candidate gene approach can follow two row in three replications. This location was also used for seed strategies (P flieger  et al  . 2001). One strategy selects tuber propagation due to low aphid pressure in this region. genes that are known to be functionally relevant for the  At Scharnhorst, 10 tubers per clone were planted in a row  trait of interest and tests their allelic polymorphisms for  without replication (Table 1). Spacing was 75 cm betweenrows and 40 cm between plants. Parental clones and eight  association with trait variation. Finding an association commercial cultivars were included as standards in each trial. between a candidate gene and the trait may indicate a To minimize border effects, the first and last plants in each causal role in trait variation (L ander and S chork 1994; row were excluded from phenotypic analysis and the trials P rioul  et al  . 1999). The second strategy compares map  were surrounded by a guard row of potato plants. positions of candidate genes with positions of QTL for  In the 1997 trials, both populations were grown at threedifferent locations: Carolinensiel and Cologne in Germany  the trait of interest to determine if they map to the and Vitoria in Spain. Seed tubers came from the Carolinensiel samegenomicregion.Furtherstudiesarethenrequired field in 1996. One-row plots with six tubers per genotype were such as linkage disequilibrium mapping (L ander  and planted in a completely randomized block design with three S chork 1994; T albot et al.  1999; M euwissen and G od-  replications at all locations (Table 1). Other than that, field dard  2000), QTL analysis of transcript and protein lev-  trials were conducted as in 1996. Depending on year andlocation, between 109 and 144 genotypes of population H94A  els (C ausse  et al.  1995), and, finally, complementation and between 126 and 171 genotypes of the H94C population analysis to validate the causal role of a candidate gene.  were evaluated in the field (Table 2). The cold-sweetening trait is particularly suitable for Tuber storage and measurement of glucose, fructose, and testing the feasibility of the candidate gene approach  sucrose content:  Potato tubers were harvested and stored at  inplants.Themetabolicpathwaysinvolvedinsugarmetab-  4   for 3 months. After cold storage, three to four randomtubers per genotype were washed, peeled, freeze dried, and olismarelimitedandwellknown.Carbohydratemetabo- ground to a fine powder. Sugars were extracted from 100 mg lism has been thoroughly studied in potato and many  dry powderaccording toV  iola andD avies (1992)with minor genes have been cloned and characterized (reviewed in modifications. Glucose (G), fructose (F), and sucrose (S) con- F rommer  and S onnewald  1995; S titt  and S onnewald  tent were measured by a coupled enzymatic assay (Boehringer 1995). Major enzymatic reactions involved in the forma-  Mannheim, Mannheim, Germany) following the supplier’sinstructions.Sugarcontentofsampleswasdeterminedbymea- tion of glucose, fructose, and sucrose in potato tubers suring NAPD reduction spectrophotometrically at 340 nm us- are outlined in Figure 1. Moreover, a potato molecular ing a microtiter plate reader (Labsystems, Germany) and the functionmapincludinggenesforcarbohydratemetabo- SCA4 software (Merlin, Germany) on the basis of standard lism and transport has been constructed (C hen  et al.  curves. Sugar content was obtained as percentage of dry tuber weight (  g/100   g dry weight). 2001). This map includes genes relevant to sugar pro-  1425Cold-Sweetening QTL in Potato TABLE 1Summary of experiments conducted for populations H94A and H94C Sugar content No. of measured afterplants percold storage b  Environment No. of clone andEnvironment code a  replications replication H94A H94CCarolinensiel 96 1 3 10    Carolinensiel 97 2 3 6    Cologne 97 3 3 6     Vitoria 97 4 3 6    Greenhouse 95 5 1 10    Scharnhorst 96 6 1 10    a  The codes assigned to each environment are used in Tables 2–4 and in the Supplementary Table. b  Sugar content corresponds to individual measurements of glucose, fructose, and sucrose content. DNA markers and map construction:  Restriction fragment analyses. All traits were analyzed separately in each of the sixenvironments.length polymorphism (RFLP) and amplified fragment lengthpolymorphism(AFLP)markerswereusedforgenotypingpop- The association between phenotype and marker genotype was investigated with both a  t  -test and interval analysis usingulations H94A and H94C. RFLP anchor markers were chosenfrom previous potato maps (G ebhardt  et al  . 1989, 2001) on SAS software (SAS  Institute  1990). Results from both meth-ods were in good agreement and, therefore, only results fromthe basis of polymorphism between parents and map position.Populations H94A and H94C were genotyped with 42 and 29 the single marker analysis are reported.  P     0.01 was theexclusion threshold for declaring the presence of a QTLRFLP anchor markers, respectively, 23 of which were mappedinbothpopulations.TheRFLPmarkersincludedthefollowing linked to a marker locus. In most cases, a QTL was detectedatseveral,closelylinkedmarkers.Toaccountforthevariability genes related to starch and hexose metabolism or transport: ADP-glucose pyrophosphorylase S and B ( AGPaseS  ,  AGPaseB  ), of QTL position due to mapping uncertainty, putative QTL were allocated to map sections (“bins”) on the basis of thestarch branching enzyme I ( SbeI  ), glyceraldehyde 3-phosphatedehydrogenase( GapC  ),apoplasticinvertase( Inv  ap  ),solubleinor- two most distal significant marker loci when considering allenvironments. The size of the bins is shown in Table 3 andganic pyrophosphatase 1 ( Ppa1 ), sucrose transporter 1 ( Sut1 ),and a putative sucrose sensor ( Sut2  ). In addition, population in Figures 2 and 3. Analysis of variance was performed at single-marker lociH94A was genotyped with cleaved amplified polymorphic se-quence (CAPS) markers for sucrose phosphate synthase ( Sps  ) among the two or four phenotypic means, depending on thenumber of marker genotypic classes distinguishable at eachand sucrose synthase 3 ( Sus3  ; C hen  et al.  2001). RFLP analysis was done as described before (G ebhardt  et al.  1989). marker locus (R  itter  et al  . 1990; S cha¨fer -P regl  et al  . 1998),using the GLM procedure of SAS. The proportion of theToincreasegenomecoverage,AFLPanalysiswasperformedaccording to V  os  et al.  (1995) with  Hin  dIII/ Mse  I and  Eco  RI/ observed phenotypic variance attributable to a particular QTL was estimated by the coefficient of determination ( R  2 ) from Mse  I adaptors. The same nine primer combinations with ex-tensions of three nucleotides (E    3, H    3, and M    3 a linear model analysis.Chi-square goodness-of-fit tests were used to test single-primers)wereusedfortheselectiveamplificationoffragmentsin both populations. The  Hin  dIII/ Mse  I primer combinations marker segregation against the expected 1:1 or 3:1 ratios. A statistical test for overlapping by chance between QTL were H    AAT combined with either M    ACA (HM1) orM    ACC (HM2) and H    ACC combined with M    AAT for the same trait in different environments was conductedfollowing the procedure of G rube  et al  . (2000). We assumed(HM3) or M   ACA (HM4) or M   ACT (HM5). The  Eco  RI/ Mse  I primer combinations were E   AAC combined with M   that for each parental map and each environment, the QTL were independent and occupied single map bins. On the basisCAG (EM1), E   ACA with M  CAT (EM2), and E   ACTcombined with either M  CAA (EM3) or M  CAT (EM4). of an average length of 750 cM of the four parental maps andthe average size of the map section covered by a QTL (TableDesignation of AFLP markers was based on the primer combi-nation and on an arbitrary identification number assigned to 3), the map was divided into 50 equal bins of 15 cM length.The probability that QTL for one trait are found by chanceeach individual AFLP fragment. AFLP markers segregating inpopulations H94A and H94C were numbered independent in the same bins in any two environments was calculated,using the hypergeometric probability distribution (B ain  andfrom each other. No attempt was made to identify identical AFLP fragments in the two populations. E ngelhardt  1992). Probabilities associated with coincidencesin three or more environments were computed in the sameLinkage maps for the 24 chromosomes of diploid potato(2 n   2 x   24)wereconstructedforeachmappingpopulation way, taking into account only conditional probability rules. Inan analogous manner, probabilities of overlaps by chanceas previously described (R  itter  et al  . 1990; S cha¨fer -P regl  et al  . 1998). between QTL for different traits across environments andbetween QTL and candidate genes were estimated. StatisticalandQTLanalyses:  Asubsetof171and188markerfragments was selected for QTL mapping, covering most of thegeneticmapsofpopulationsH94AandH94C,respectively.The phenotypic values for the traits glucose, fructose, and RESULTSsucrose content per line in six environments (years and loca- Evaluation of sugar content:  Tuber sugar content  tions, Table 1) were obtained as means of three to four tubersper replication. These mean values were used in the QTL  after cold storage was evaluated in populations H94A   1426 C. M. Mene´ndez  et al. TABLE 2Statistical parameters of sugar content after cold storage and number of clones ( N   ) analyzedfor populations H94A and H94C H94A H94CEnvironment Sugar Range of Sugar Range of Trait code a  N   content  b   variation  N   content  b   variationGlucose 1 144 1.56 (0.81) 0.20–4.39 147 0.94 (0.51) 0.11–2.452 109 1.08 (0.63) 0.09–3.03 126 1.16 (0.52) 0.25–2.503 132 0.97 (0.55) 0.07–3.33 137 0.84 (0.45) 0.07–2.104 117 0.80 (0.58) 0.01–2.79 153 0.88 (0.43) 0.02–2.455 132 0.39 (0.39) 0.01–2.22 169 0.42 (0.37) 0.01–1.936 125 0.90 (0.73) 0.06–3.75 153 0.55 (0.48) 0.05–3.60Fructose 1 144 1.48 (0.69) 0.24–3.84 147 1.02 (0.49) 0.04–2.542 109 1.17 (0.57) 0.22–2.92 126 1.35 (0.47) 0.28–2.533 132 1.28 (0.62) 0.24–3.29 137 1.30 (0.55) 0.20–3.014 117 1.14 (0.58) 0.07–3.16 153 1.17 (0.46) 0.06–2.405 132 0.54 (0.45) 0.01–2.45 169 0.62 (0.41) 0.03–1.796 125 1.25 (0.78) 0.13–4.15 153 0.82 (0.56) 0.14–3.55Sucrose 1 143 1.67 (0.81) 0.44–4.51 147 1.29 (0.64) 0.52–4.162 109 2.84 (1.62) 0.63–7.21 126 1.89 (1.02) 0.61–6.413 132 1.33 (0.81) 0.32–4.56 137 1.17 (0.51) 0.5–3.884 117 1.30 (0.81) 0.41–7.22 153 1.99 (1.06) 0.51–7.055 126 0.97 (0.57) 0.23–4.47 169 0.73 (0.25) 0.25–2.606 122 0.91 (0.49) 0.20–2.51 153 0.79 (0.33) 0.05–2.29 a  Environment codes are as shown in Table 1. b   Valuescorrespondtoaveragesugarcontent(percentagedryweight).Standarddeviationsareinparentheses. and H94C over 3 years at four locations, resulting in six 0.77. Correlations in sugar content of tubers grown inpots and in the field varied from 0.53 to 0.70 for H94A environments (Table 1). Table 2 shows populationmeans,standarddeviations,andrangesofsugarcontent and between 0.19 and 0.35 for H94C. Maps of populations H94A and H94C:  Twenty-four(percentage dry weight) of glucose, fructose, and sucrose.Reducing sugar content was lower in greenhouse-grown linkage groups, 12 for each parent, were constructedfor populations H94A and H94C on the basis of 433tubers than in the field (Table 2, environment 5).Sugar contents were approximately normally distrib- and 447 RFLP and AFLP marker fragments, respectively (Figures 2 and 3). The level of heterozygosity was highuted in the populations and showed transgressive segre-gation in all environments (not shown). Based on the in the parents. Only clone P54 (P  A  of population H94A)appeared to be less heterozygous than the other paren-ranges observedfor sugarcontents, lessphenotypic vari-ability was present in population H94C when compared tal lines, on the basis of the smaller number of segregat-ing fragments descending from that parent. Marker dis-to H94A (Table 2). Glucose and fructose contents of tubers after cold storage were highly correlated in all tribution on the linkage groups was uneven, mainly dueto clustering of AFLP markers. Genome coverage wasenvironments, with phenotypic correlations ranging from0.89 to 0.93. Correlations in sugar content of tubers incomplete in the H94A map in regions of linkage groupsIA, IIA, VIA, and XIIA of parent P54 (P  A  ) and IB, IVB,grown in different field environments were lower, but still highly significant with values ranging from 0.50 to and IXB of parent P40 (P B ). In the H94C map, gaps  Figure  2.—QTL and linkage maps of population H94A. Linkage groups A and B are derived from the parents P54 and P40,respectively. Allelic bridges (R  itter  et al.  1990) linking the parental linkage groups are not shown. HM*/* and EM*/* are AFLPmarkers obtained with  Hin  dIII/ Mse  I and  Eco  RI/ Mse  I primer combinations, respectively. CP* and GP* are RFLP markers. Genemarkers (further details in C hen  et al.  2001; G ebhardt  et al.  2001) included are ADP-glucose pyrophosphorylase S and B( AGPaseS  ,  AGPaseB  ), 4-coumarate: CoA ligase ( 4Cl  ),  ocs  -like bZIP-binding element ( mbf    ), starch-branching enzyme I ( SbeI  ),putativesucrosesensor( Sut2  ),glyceraldehyde3-phosphatedehydrogenase( GapC  ),wound-inducedgenes1and2( WUN1 , WUN2  ),phosphoenolpyruvate carboxylase ( Ppc  ), sucrose phosphate synthase ( Sps  ), sucrose synthase 3 ( Sus3  ), pyrophosphatase 1 ( Ppa1 ),lipoxygenase ( Lox  ), apoplastic invertase ( Inv-ap  ), and sucrose transporter 1 ( Sut1 ). Loci detected by genes functional in starchand hexose metabolism or transport are shaded gray. Candidate genes  Pain-1  and  UGPase   were mapped in a different population(C hen  et al  . 2001) and are shown at their approximate positions. Lowercase letters in parentheses indicate that more than onelocus was detected with the same marker probe. Linkage group regions where markers were significant in the  t  -test are indicatedby dotted lines and labeled with QTL names according to Table 3.  1427Cold-Sweetening QTL in Potato
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