Molecular Marker Linkage Map for Apple

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Molecular Marker Linkage Map for AppleM. Hemmat, N. F. Weeden, A. G. Manganaris, and D. M. Lawson

Linkage maps for two apple clones, White Angel and Rome Beauty, were constructedusing isozyme and DNA polymorphisms segregating in a population produced froma Rome Beauty x White Angel cross. The linkage map for White Angel consists of253 markers arranged in 24 linkage groups and extends over 950 cM. The RomeBeauty map contains 156 markers on 21 linkage groups. The White Angel map wastaken as the standard, and we were able to identify linkage groups in Rome Beautyhomologous to 13 White Angel linkage groups. The location of several genes notsegregating in the Rome Beauty x White Angel population could be determined onthe basis of known linkages with segregating markers. Hence, the standard map forapple now contains about 360 markers, with most linkage groups saturated at 10-15 cM. The double pseudotestcross format of the mapping population permitted thecomparison of recombination frequencies in male and female parents in certainregions of the genome where appropriate markers were available. The recombinationfrequencies observed for the approximately 170 cM that were comparable gave noindication that a sex-related difference in recombination rate was characteristic oapple.

From the Department of Horticultural Sciences, Cor-nell University. Geneva, New York 14456 (Hemmat,Weeden, and Lawson) and the National AgriculturalResearch Foundation. Pomology Institute, 59200Naoussa, Greece (Manganaris). We thank Susan Gar-diner and Susan Brown for helpful suggestions on themanuscript.Journal of Heredity 1994;85:4-11; 0022-1503/94/15.00

The domesticated apple (Malus x domesticsBorkh.) is not commonly used in moleculargenetic studies. The long generation time,large plant size, high chromosome number(2n = 34), and self incompatibility typical ofthis species have made apple, as well asmany other woody perennials, less appeal-ing to geneticists than species such as maize,tomato, and Arabidopsis thaliana. Yet linkagemaps and molecular tags for charactersexpressed late in development would beparticularly useful in breeding programsfor long-lived perennial crops. Earlyscreening for fruit quality traits, precocity,or growth habit would permit much moreefficient use of available orchard space,and marker-based cloning of specific geneswould greatly enhance our ability to makeminor changes in popular varieties.

Many woody species exhibit high levelsof genetic polymorphism (Hamrick andGodt 1990), and in outcrossing species thishigh polymorphism usually produces ahigh level of heterozygosity in individualplants. Thus, many loci will be segregatingin the gametic population from a singleplant, and if these gametes can be exam-ined individually a linkage map can be eas-ily generated. Conifers such as pines andfirs produce a relatively large megaga-

metophyte. This haploid tissue has pro-vided an excellent tool for genetic inves-tigations of isozyme loci (Cheliak et al.1984; Eckert et al. 1981; El-Kassaby et al.1982; Guries et al. 1978; O'Malley et al.1979), restriction fragment length poly-morphisms (RFLPs) (Neale and Williams1991), and random amplified polymorphicDN A (RAPDs) (Carlson et al. 1991). Link-age maps have been constructed for singletrees of loblolly pine (GCrattapaglia et al.1992) and white spruce (Tulsieram et al.1992) by a thorough study of the loci seg-regating in the megagametophytes.

Angiosperms rarely produce gameto-phytes that are large enough to providesufficient tissue for multilocus studies. In-stead, backcross, testcross, or F2 popula-tions generally have been selected to examine the segregation of gametes from aheterozygous individual. Commonly, theheterozygous individual is generated as ahybrid between two relatively homozy-gous lines. However, in apples and manyother outcrossing species, this initial stepis unnecessary. Each apple variety is highly heterozygous due to the clonal natureof the crop and the poor performance as-sociated with inbred material. Inbreedingdepression also precludes the regular use

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of backcross or testcross populations forgenetic analysis because inbred lines arenot available. Instead, a pseudotestcrossdesign is typically used in which the va -riety of interest is crossed to a standardvariety known not to segregate for the traitsbeing investigated. The segregation ratiofor single gene traits is 1:1 in such a pop-ulation, and genetic analyses can be rel-atively simple.

A refinement of this approach is to per-form genetic analysis on both parents ina controlled cross by keeping track ofwhich loci are heterozygous in each par-ent. Such a "double pseudotestcross" for-mat represents the basic experimental de-sign in most apple breeding programs andhas been used to identify linkage relation-ships among isozyme loci and betweenisozyme loci and other traits of practicalinterest to breeders (Chevreau and Lau-rens 1987; Manganaris and Alston 1987,1988a,b; Weeden and Lamb 1987). In theresearch reported here we expanded thetype of markers used in apple to includeboth DN A restriction fragment lengthpolymorphisms (RFLPs) and random am -plified polymorphic DN A (RAPDs) in or-der to generate a relatively saturated ge-netic map for each parent. We thenattempted to combine he two maps intoa more general linkage map for the spe-cies.

Materials and MethodsPlant MaterialWe used data collected from 56 trees pro-duced from a Rome Beauty x White Angelcross to construct the linkage map. Thecross had been made in 1986 by R. C. Lamb,Department of Horticultural Sciences, Cor-nell University. The seeds were germinat-ed the following year after treatment at 5Cfor 90 days to break dormancy. Survival ofseedlings was excellent, so that the prog-eny represented a relatively unbiasedsample of the original seed population.Rome Beauty is a commonly cultivated ap-ple originally found in a fence row in Ohioin 1848 (Beach et al. 1905). White Angelis a crabapple of unknown parentage dis-covered in Inglis nursery about 1947 (Jef-ferson 1970). Analysis of its 45S ribosomalDNA restriction pattern suggests that it hasMalus seiboldii in its ancestry (Simon andWeeden 1991). White Angel is believed tobe heterozygous for a dominant gene con-ferring resistance to powdery mildew(Podosphaera leucotricha)(Manganaris andAlston 1992), whereas Rome Beauty ishighly susceptible.

Isozyme SystemsExcept where noted, we performed iso-zyme analysis on young leaves collectedin the spring. Analyses of polymorphismfo r aspartate aminotransferase (AAT; EC2.6.1.1), (t acid phosphatase (ACP, EC3.1.3.2), endopeptidase (ENP, EC 3.4.9.9),fluorescent esterase (EST, EC 3.1.1.-), glu-cosephosphate isomerase (GPI, EC 5.3.1.9),isocitrate dehydrogenase (IDH, EC1.1.1.41), leucine aminopeptidase (LAP, EC3.4.11.1), malate dehydrogenase (MDH, EC1.1.1.37), malic enzyme (ME, EC 1.1.1.40),6-phosphogluconate dehydrogenase (PGD,EC 1.1.1.44), phosphoglucomutase (PGM,EC 5.4.2.2), peroxidase (PRX, EC 1.11.1.7),superoxide dismutase (SOD, EC 1.15.1.1),and triose phosphate isomerase (TPI, EC5.3.1.1) were performed on horizontalstarch gels or vertical polyacrylamide gelsas described previously (Manganaris andAlston 1987; Weeden and Lamb 1985,1987). Alcohol dehydrogenase (ADH, EC1.1.1.1) was performed on cambial scrap-ings as described in Hagens (1992). Usinghorizontal starch gels we also resolved en-zyme systems not described previously inapple. These systems were alanine ami-notransferase (ALAT, EC 2.6.1.2), shikim-ate dehydrogenase (SKDH, EC 1.1.1.25),glutamate dehydrogenase (GDH, EC1.4.1.2), formate dehydrogenase (FDH, EC1.2.1.2), and aconitase (ACO, EC 4.2.1.3).Cambial scrapings were extracted for FDHanalysis. Assay conditions for these newenzyme systems were as described inWendel and Weeden (1989).We extracted high molecular weight DNAappropriate for RFLP analysis from ex -panding apple leaves essentially as de-scribed in Polans et al. (1985). This DNA(10-15 Mg per sample) was restricted andsubjected to Southern analysis as de-scribed in Weeden et al. (1992). The plas-mids used were pHA2, containing the pea45S RNA repeat (Jorgensen et al. 1987),plasmids kAt3011 and jAt3012 containingthe 3' and 5' portions of the ADH sequencefrom Arabidopsis thaliana (kindly provid-ed by E. M. Meyerowitz, California Instituteof Technology), and four random clones(pAP79, pAP236, pAP244, and pAP260)from an apple EcoRI genomic library inpUC13. Parental DNA was restricted withEcoRI, EcoRV, Hindll, BarnHIl, BgIlll, Xbal,and Dral in an initial screen to determinewhich enzymes generated fragments ap-propriate for analysis.We extracted DN A for RAPD analysisfrom small amounts (ca 0.5 g) of youngleaf tissue by a modification of the pro-cedure of Lassner et al. (1989) as de-

scribed in Torres et al. (1993). ExtractedDNA was diluted in autoclaved distilledwater to a concentration of about 25 ug/ml and subjected to amplification on a Co yModel 50 TempCycler. Each 25-1l PCRmixture contained 2 l template DNA, 13.2l H20, 2.5 l 10 x buffer, 1.25 ul of 2.5 mMof each dNTP, 0.4 ,1 of 20-,M primer, and0.5 units Promega Taq polymerase. Cy-

cling parameters were 40 cycles (94C, 1min; 35C, 2 min; 72C; 2 min) followed bya 6-min extension at 72C. Primers weresynthesized at the Cornell University Bio-technology Institute or purchased fromGenosys Biotechnologies, Houston, Texas.Products of the amplification reaction wereseparated on 2% gels (1% agarose and 1%NuSieve, FMC Corporation) run for 4 h at100 V in I x TB E buffer (Sambrook et al.1989). We stained gels with ethidium bro-mide and photographed them on a trans-illuminator (X = 302 nm) using PolaroidType 55 film; segregation patterns werescored off the negatives. Scoring was al-ways done on the basis of a "fragmentpresent"/fragment absent" dichotomy.Powdery Mildew ResistanceWe scored resistance to powdery mildewin the field over a period of 4 years. Thepresence of mildew on the leaves andyoung branches was considered evidenceof susceptibility. Plants never showing suchinfestations were scored as resistant.Linkage AnalysisWe divided markers into three groups: (1)those segregating as a result of heterozy-gosity in White Angel; (2) those segregat-ing as a result of heterozygosity in RomeBeauty; and (3) those segregating as a re-sult of heterozygosity in both parents. Foranalysis of amplified DN A fragments, weconsidered a parent heterozygous for aparticular segregating fragment if DNAfrom that parent generated the fragmentwhen used as template DNA. The expectedsegregation ratio for a locus heterozygousin only one parent was 1:1, whereas locheterozygous in both parents were ex-pected to display either a 3:1 ratio for dom-inant characters such as RAPDs or a 1:2:1ratio for codominant allozymes and RFLPs.Goodness-of-fit between observed and ex-pected segregation ratios was tested usingchi-square analysis as performed byLINKAGE-I (Suiter et al. 1983).

Two linkage maps were generated, onewith those loci heterozygous only in WhiteAngel and the other with loci heterozy-gous only in Rome Beauty. For each of theWhite Angel and Rome Beauty data sets,

Hemmat et al Linkage Map of Apple


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we performed initial joint segregationanalysis on about 60 loci using LINKAGE-1. Once preliminary linkage groups hadbeen identified, we used QUIKMAP (an Ex-cel spreadsheet operation available fromN.F.W.) to ma p additional markers. MAP-MAKER (Lander et al. 1987) was used toconfirm locus order for each linkage groupand to determine multipoint recombina-tion frequencies among loci. A log-likeli-hood score of 3.0 and a recombination dis-tance of no more than 20 cM were theminimal criteria for establishing linkagebetween markers. The "ripple" functionon MAPMAKER was employed to assessthe robustness of the order of markers ona linkage group. In general, we specifiedan order only when no alternative markerorder with an LOD within -3.0 was avail-able. After the maps had been assembled,we used QUIKMAP to check for the exis-tence of significant linkages betweenmarkers in different linkage groups.Th e White Angel an d Rome Beauty mapswere compared, and we identified corre-sponding linkage groups using loci het-erozygous in both parents (for detailed de-scription of this technique see Hagens 1992;Lawson DM, Hemmat M,and Weeden NE,unpublished manuscript). Only data forwhich the contribution of each parent wasunambiguous were used for mapping lociheterozygous in both parents; hence, onlythe homozygous genotypes were used tomap isozyme and RFLP markers. For RAPDmarkers, heterozygotes could no t be dis-tinguished from the homozygous 'frag-ment-present' phenotype, so only the ho-mozygous recessive (fragment-absent)genotypes were used for this analysis.ResultsWe identified 409 segregating markers inthe population. A complete list of thesemarkers is available upon request and isposted on the Internet Gopher (copyright1991, University of Minnesota) under Cor-nell University, College of Agriculture, Ge-neva, Department of Horticultural Sciences,Fruit Breeding and Genetics. Thirty-four ofthese markers were allozyme polymor-phisms, eight were RFLPs, and the remain-der were amplified DNA fragments gener-ated by arbitrary primers. Most of themarkers displayed segregation ratios no tsignificantly different from the 1:1, 1:2:1,or 3:1 ratio expected based on parentalphenotypes. White Angel displayed het-erozygosity for 268 markers, and RomeBeauty wa s heterozygous for 180 markers.Of these, 39 loci were heterozygous in both

parents or , in the case of RAPDs wherehomology could not be confirmed, be-haved as such by being present in bothparents and segregating in the progeny.Segregation patterns for allozyme poly-morphism for AAT, ACP, ADH, DIA, EST,GPI, IDH, LAP, MDH, ME, PGD, PGM, PRX,SOD, and TPI were defined as describedpreviously (Hagens 1992; Manganaris 1989;Weeden and Lamb 1985). Polymorphismin ALAT, ACO, FDH, GDH, and SKDH willbe described in a separate paper in whicha wider sample of germplasm is analyzedand discussed. For ALAT, FDH, GDH, andSKDH only a single polymorphism couldbe observed, although at least on e addi-tional isozyme was present in each sys-tem. For ACO, three polymorphic regionson the gel were identified. These were la-beled ACO-I, ACO-2, and ACO-3 in orderfrom the most anodal region. In otal, WhiteAngel displayed heterozygosity at 24 iso-zyme loci an d Rome Beauty at 14 .Th e RFLP patterns generated by theprobes were all multibanded, usually dis-playing one or more monomorphic frag-ments in addition to those polymorphic.For the mapping analysis we used EcoRlphenotypes for pHA2, Dral for pAP260,EcoRV for pAP236, and BamHl for pAP79and pAP244. The ADH probes revealedpolymorphism with several restriction en-zymes, with Hindlll exposing a polymor-phism for Adh-2heterozygous in both par-ents (Hagens 1992). The Adh-2 RFLPheterozygous in White Angel cosegregatedwith the allozyme variation, indicating thatthe two were synonymous.Th e greatest number of heterozygousmarkers were identified with arbitraryprimers. Sixty-four primers or primer com-binations were used in the analysis (Table1). On average, 10 well-defined fragmentswere generated by each primer, about halfof which were polymorphic (Figure 1).Several primers'(e.g., P1, P5, PIO, an d P21)generated relatively few products. Certainpairwise combinations of these primers(e.g., P and P5, and P2 and P10) gener-ated novel fragments and polymorphismsno t produced when either of the primerswas used individually.Although duplicate samples amplifiedon different days using the same primergave very similar phenotypes, some vari-ation was noted, particularly in the lessstrongly amplified products (fainterbands). Overall, we found a "misscoring"or "irreproducibility" rate of between zeroan d 7%, depending on the fragment beinganalyzed (Weeden et al. 1992). These rateswere determined not only by replicate

Table 1. Primers used for producing RAPDa onapple linkage mapPrimerdesignation Sequence (5'-3')TI AGT TCG TCTGT2 TGG TGG GTC CT3 CTC GGT ACA CT4 GTG GTT GCG AT5 CTG GAC TTA CP1 CCT GTA GTGGP2 TAC CTT CCG TP3 GTC CGT TGG GP4 GTT AGG TCG TPS TCT CTG TCC CP6 TCG CCC CAT TP7 CGT GGT TCC CP8 GTC CCG TTA CP9 ACG CCC TAG TP10 TT CAC ATG GPIl CTG TGC TGT GP12 TGG TGG ATG TAP1 3 GGT AT GTC CP14 TGC CTT CCA TP15 GGG TTG CCGTP17 CTA TTT TGC CP21 CCC TGT CTC TP24 AGC ACT GTC AP2 5 AAT GAA GCC AP2 6 TAC TGC TGG GP2 7 ACC TCG AGC AP2 8 TCG TAG CCA AP3 0 CAA CTG GTA ATGP9 8 GAA CGA CGC AP105 CAG TCG CGT GPI 17 GGG ATCGTG TP123 GGG ATT CGA CP124 ACT ACT GCC TP126 CTG CTA CGT CP129 CAG TCG AAC GP137 ATC TGC GAC AP145 TAG CGG CTA CP157 GTC ATG TCG AP161 CGG ATG CCT TP163 ACG CCT ACG TS2 CGA GTT GCG CS5 CCG GCT CTT GS12 GCG ACG CCT AS15 AAC ACA TGC C$16 CGT TGG ATG CS18 TTT GG C TCT GS19 TAC GGC TGG CS20 TGA ACC GCC GS21 CCC TAC CGA CS22 CGT CGT GGA AS23 CCA CGC TAT AS24 GCG GCA TTG TS27 AGT GGT CGC GS3 0 GCG TAG AGA CS31 CTC GAC ACTGS34 GAT AGC CGA CS35 AGT CGC TCA TS36 CTC CAA GGC CS39 TCG GCC TGC TS40 GAC TGC TCG GS42 CCC AGA ACA CS44 ATT CGG CCG GS45 CAC GTC GGA GS4 9 ACC GGG CCT AS50 CCC AAA CTA G

RAPD analyses but also by comparisonwith tightly linked isozyme markers (whichhad been scored several times) and bydetailed analysis of clustered RAPD markers. For instance, the markers P123j, P24gT4a, P3k, P137g, and S42i all gave nearlyidentical segregation patterns. Th e few re-combinants identified among these mark

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to incorporate either into one of the otherpaired linkage groups identified.

Figure 1. RAPD phenotypes obtained on parental and progeny template DNA for three primers (S42, S30, andS36). Five apple samples are shown for S42 and S30, and lour are shown for S36. Lanes containing an Hoell cutx 174/Hindlll cut X combination size marker separate the sets. The sequence of samples within each set is(from left) three individuals from the F, population, Rome Beauty, and White Angel. The White Angel phenotypeis not shown for S36; however, the positions of the five S36 RAPDs are marked to the right of the figure by theirrespective letter designations. The sizes of the marker fragments (in kbp) are given on the left of the figure.

ers did not assort themselves into a linearsequence, and the majority thus became"double recombinants" within the cluster,suggesting that these data are actuallyerrors in amplification/scoring. We felt themost reliable way to present the linkagerelationship among such loci was to dis-regard (i.e., treat as missing data) theresults generating double recombinantswithin such clusters. This option causedsuch clusters to condense and shortenedthe linkage map around these regions.However, new linkages were not formed,nor were significant alterations in markersequence produced by this operation.Linkage AnalysisThe White Angel map possessed 253 mark-ers arranged on 24 linkage groups, cov-ering 950 cM (Figure 2). Five markers, het-erozygous only in White Angel, could notbe placed on the map. Results for RomeBeauty were similar except that consid-erably fewer (156) markers were mappedand 14 markers assorted independently(Figure 2). In addition, 10 RAPDs that ap-peared to be heterozygous in both parentscould not be located on the linkage map.The map for White Angel was chosen asthe standard map for additional compar-isons and analysis because it includedmore markers. All linkage groups in bothparents were found to be well defined.Markers on one linkage group did not dis-play significant linkage (LOD > 3.0, recom-bination fraction 25 cM ) between markers on

different linkage groups were identified.However, the large number of markersbeing compared made it likely that suchlinkages would be identified in a group ofrandomly assorting markers, and we dis-regarded these correlations when assem-bling our groups.

Homologous counterparts to 15 of theWhite Angel linkage groups were identi-fied on the Rome Beauty map using mark-ers heterozygous in both parents (Figure2) . For six linkage groups (3, 5, 6, 7, 9, and10) the homology was confirmed by twoor more markers, permitting the homol-ogous linkage groups to be placed in thesame orientation relative to each other.Rome Beauty counterparts to the relative-ly long White Angel linkage groups 1, 2,and 8 could not be identified. Conversely,three long (>30 cM ) linkage groups ofRome Beauty were not paired with WhiteAngel linkage groups through loci hetero-zygous in both parents. However, previousstudies on linkage relationships amongisozyme loci in apple (Manganaris 1989;Weeden and Lamb 1987) suggest that theRome Beauty linkage group containingPgm-I and Tpi-c2 was not homologous tolinkage groups 1 or 2 of White Angel. Thislinkage group is particularly important fo rbreeders (see Discussion), and we havedefined our linkage group 8 using the RomeBeauty group rather than that from WhiteAngel. At present, we have no direct evi-dence of homology between the White An -gel and Rome Beauty linkage group 8. Thetwo are paired because both are of suchlength (>60 cM ) that it would be difficult

Distribution of RAPDsApproximately 80% of the RAPDs on a par-ticular White Angel linkage group did notappear to have counterparts on the cor-responding Rome Beauty linkage group.However, 6% of the RAPDs appeared to beproduced by both parents, and at least an-other 5% of the amplified fragmentsshowed similar intensity and size in bothparents but did not segregate in the prog-eny. In addition, a primer would occasion-ally produce an RAPD in a similar locationon both maps, although the size of thefragment would differ. For instance, prim-er P4 produced an 850-bp RAPD on theWhite Angel linkage group 14 and a frag-ment of size 93 0 bp at a correspondingposition on the Rome Beauty map. A sim-ilar correlation occurred for primers S34and P123 on linkage group 16. In addition,several cases were noted where an RAPDheterozygous in both parents showed tightlinkage with another RAPD generated bythe same primer (e.g., P98a, P98b, and P98ion linkage group 9; S45c and S45f on group3; P27a and P27e on group 6; and P27c andP27d on group 14).

In most cases the separate DNA frag-ments generated by a single primer as-sorted independently; however, in severalcases such fragments demonstrated link-age or even tight clustering. For instancethe 820- and 550-bp fragments generated byS20 both mapped on White Angel chromo-some 12, although a significant distanceapart. A similar situation was observed onlinkage group 8 for the 1030- and 350-bpfragments produced by primer S15, and foP117f and j on Rome Beauty group 10. The880- and 420-bp fragments of S16 bothmapped at the end of White Angel linkagegroup 6. Several such clusters also were evident on the Rome Beauty map: P98d andP117d and I, P123d and k, and S49b and p

The most conspicuous clusters on themap contained RAPD markers generatedby different primers. One of these clusterwas found on both the White Angel andRome Beauty group 16. In White Angel thiscluster included 11 RAPD markers within10 cM, whereas in Rome Beauty six markers clustered within this distance. Smallerclusters were found on nearly all linkagegroups.Comparison of Male and FemaleRecombinatlon FrequenciesLoci heterozygous in both parents werused to identify homologous linkage



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groups. Mapping of more than one suchlocus on a linkage group permits the directcomparison of recombination frequenciesin the male and female parents for the seg-ment of the map between the tw o loci. Suchcomparisons can be made for loci on link-age groups 3, 5, 6, 7, and 9. On linkagegroup 5, Rome Beauty exhibits higher re-combination rates between markers; how-ever, White Angel showed higher levels ofrecombination on group 6 (Table 2). Ongroups 3, 7, and 9 the average recombi-nation frequency is nearly equal for thetwo parents, although the placement ofP105e on each of the group 5 maps sug-gests that recombination frequencies forspecific segments of the maps may differbetween the parents.

Unfortunately, when a locus is hetero-zygous for the same alleles in both par-ents, the contribution of each parent isambiguous for certain phenotypes (e.g.,heterozygotes for codominant alleles andthe dominant phenotype for RAPDs andother dominant/recessive markers).Hence, only half or one-fourth of the pop-ulation gave data useful for determiningthe position of markers heterozygous inboth parents. Consequently, the locationof these markers is much less precise thanthe locations of the markers heterozygousin only one parent. In Figure 2 the posi-tions of most of the markers heterozygousin both parents are represented by a barparallel to a region of the respective link-age maps, reflecting the greater uncertain-ty associated with this position. The highuncertainty of the linkage estimates givenin Table 2 also reflects this limitation. Theexceptions to this convention-ldh-2 ongroup 3, Adh-2 on group 6, and Lap-I ongroup 18-gave segregation phenotypesin which the allelic contributions of theparents were unambiguous, and their po-sitions could be determined with the stan-dard precision.

DiscussionExtensive linkage maps for two appleclones, one a common commercial varietyand the other a crabapple with possibleM seiboldiiheritage, were generated usingallozyme and DNA polymorphisms. Thesemaps were generated in an F, populationproduced by crossing the tw o varieties andtaking advantage of the high level of het-erozygosity often present in long-lived, selfincompatible species. This high hetero-zygosity in apple has been documented byseveral investigators (Chevreau et al. 1985;Manganaris 1989; Weeden and Lamb 1987),

and many loci can be expected to segre-gate in the immediate progeny of any cross.In the present cross, White Angel exhib-ited about 50% more heterozygosity thanRome Beauty, presumably reflective of itsmore diverse parentage.

The development of the maps was great-ly facilitated by the use of short oligonu-cleotide primers to generate discrete am-plified segments of DNA as described inWilliams et al. (1990). These markers werefound to be particularly suitable for ourapproach to genetic analysis in apple. Anaverage of six polymorphisms per primerwas obtained in this study, and compa-rable levels have been observed in otherpopulations (Hemmat M, unpublisheddata). With such high levels of polymor-phisms generated by single primers, wefound little need to use primers in pairs.The small gain in resolved RAPDs whenwe combined certain primers (e.g., PI andP5 ) was not cost-effective.

The dominant nature of RAPD markersdid not represent a significant disadvan-tage in our analysis. At least fo r the casein which only one parent is heterozygous,a dominant/recessive allelic system willdisplay the same 1:1 segregation ratio asany other polymorphic locus. Hence, thedominance problem that hinders the ap-plication of RAPD markers in mappingmany annual crops, such as maize andsoybean, appears to be much less of aproblem in highly heterozygous species.Only when an RAPD fragment is hetero-zygous in both parents does the fragment-present phenotype represent an ambigu-ous genotype in the progeny. In this casethe fragment-absent phenotype is still use-ful for mapping the polymorphism, but asit occurs only in one-fourth of the progenythe amount of data available for linkagetests is greatly reduced. Such RAPDs areparticularly useful for identifying homol-ogous linkage groups between the two par-ents, and we attempted to map all that weencountered. We were successful onlyabout 60% of the time despite being ableto map over 90% of the polymorphismsheterozygous in only one parent.

The utility of RAPD markers also is be-lieved to be limited by the difficulty inidentifying homologous markers in differ-ent crosses. Our results suggest that only10%-20% of the amplified fragments werecommon to both parents. However, WhiteAngel and Rome Beauty represent widelydivergent apple germplasm. Preliminarystudies in our laboratory on a series ofcommercial apple varieties indicate thatthese share a much higher proportion of

Table 2. Comparison of map distances betweensyntenic markers mapped on both the RomeBeauty and White Angel linkage groups

Linkagegroup Markerscalculated distanceRome WhiteBeauty Angel

3 ldh-2 :S40d 39 12 39 125 S5g : S45d 34 + 16 26 + 156 S15f :Adh-2 38 + 14 42 + 177 S22d Aat-p 20 t 20 22 109 S45a S16d 17 11 18 + 14

amplified fragments. The recent use oRAPDs to fingerprint apple varieties byKoller et al. (1993) also indicates that majority of the amplified products are con-served among many commercial varieties.More important, apple breeders often usea limited number of parents repeatedly intheir breeding programs. For instance, thevariety Golden Delicious has often beenused as a parent because it consistentlyproduces progeny of commercial quality.RAPD markers identified in such impor-tant varieties are likely to be useful in manybreeding programs. Thus, the limitationsassociated with the use of RAPDs in thegenetic analysis of annual crops or wildpopulations are at least partially mitigatedby characteristics and breeding tech-niques peculiar to longer lived crops.

In most cases, the White Angel and RomeBeauty maps were consistent with previ-ous findings (Manganaris 1989; Weedenand Lamb 1987). Aat-c was tightly linkedto Idh-I on linkage group 1; Acp-l, Enp-land the modifier of PGM phenotype, Pgmr, mapped near each other on linkage group4; and powdery mildew resistance, P,showed linkage to Acp-3 and Aat-p as wasreported by Manganaris (1989), althougha more tightly linked isozyme marker, Lap-2could not be resolved in our cross. Man-ganaris (1989) found PGD-3to be linkedto dh-l. Our locus Pgd-c2, positioned onthe short linkage group 17, may be thesame as PGD-3. However, markers on theWhite Angel group 17 assorted indepen-dently of markers on linkage group 1.Man-ganaris also found linkage between Lap-and Pgm-2.Our data could not confirm thislinkage, but the intermediate isozyme lo -cus, Aat-4 (=GOT-4) was not segregatingin our population. If the two loci are onthe same linkage group, group 18 can beassigned to the lower end of group 9. Wewere not able to confirm the Pgm-2-Adh-2linkage reported by Manganaris (1989). Inour cross, Adh-2 cosegregated with mark-ers on linkage group 6, whereas Pgm-2displayed linkage with markers on group 9



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inconsistencies may reflect chro-rearrangement known to bein the apple germplasm (Zhu MS,personal communication).Several markers not segregating in ei-could be located on the mapon previously determined linkages.are given in parentheses at theirrespective positions in Figure 2. They in-clude the self incompatibility locus, S. onlinkage group 1 (Manganaris an d Alston

1987); Lap-3 and Mdh-I on group 2 (Man-ganaris 1989); Dia-2 on group 4 (Weedenand Lamb 1987); Pgm-4, PI,, and Lap-2ongroup 7 (Manganaris 1989); and Vh Dia-5,and Mdh-2 on group 8 (Manganaris 1989;Weeden and Lamb 1987). Th e loci S, V,PI,, and Pi, are particularly important forapple breeders as they govern self incom-patibility, scrab resistance, and powderymildew resistance, and it is crucial to havetheir locations on the map well docu-mented.The combination of the two maps usingmarkers heterozygous in both parents rep-resents a specialized application of genet-ic principles that is appropriate when gen-erating maps in such outcrossing species.Codominant loci were particularly usefulin this application because, theoretically,half of the progeny (those homozygous foreither allele) give information useful formapping. Hagens (1992) used isozymesand RFLPs to convincingly demonstratethe homology between linkage groups 6 ofWhite Angel and Rome Beauty, and theisozyme loci Idh-2 and Aat-p clearly estab-lished linkage groups 3 an d 7, respective-ly, in both parents. In contrast, for domi-nant/recessive markers, only a quarter ofthe progeny (those homozgyous for re-cessive allele) give useful information foridentifying homologous chromosomes.Several of the RAPDs heterozygous in bothparents could not identify homologousgroups because the amount of informativedata was too small, and homologies estab-lished on the basis of a single RAPD mark-er (e.g., groups 11-17) must be acceptedwith a degree of caution.On linkage groups where more than onelocus heterozygous in both parents weremapped, a comparison of the recombi-nation frequencies producing male and fe-male gametes could be made. Althoughseveral studies have indicated a lower lev-el of recombination rates in male versusfemale gametogenesis (de Vicente andTanksley 1991; Rick 1969, 1972; Robertson1984), our results indicate approximatelyequal recombination frequencies for thetwo parents. Our results could not account

for possible effects of the different geneticbackground in the two parents, and theless precise mapping of the doubly het-erozygous loci used for the comparisonalso may have obscured a small consistentdifference in recombination frequency.Thus, our conclusion that the data do notsuggest a lower recombination level inWhite Angel must be considered prelimi-nary. A more complete survey of the applegenome using reciprocal crosses is inprogress.A particular concern of many research-ers working with RAPD markers is the re-liability and reproducibility of this tech-nique. We observed an error frequency ofabout 5% n scoring RAPD markers in pea(Weeden et al. 1992) and anticipated thatou r RAPD data in apple also would showthis level of inconsistency. We attemptedto eliminate or correct for such errors byusing a DNA extraction procedure thatgives small amounts of very high qualityDNA, by scoring only very clear polymor-phisms, and by anchoring most linkagegroups with allozyme or restriction frag-ment length polymorphisms. These mea-sures appear to have been successful be-cause internal controls and data analysisestimated only a 2% error in ou r RAPDdata (Weeden et al. 1992). Most of the clearRAPDs could be easily and confidentlymapped with approximately the numberof double crossovers expected from thedistances involved. Concordance of seg-regation data in clusters of nonrelatedRAPD markers also demonstrated the gen-eral accuracy of the data. In cases whererecombination was observed between anRAPD fragment and another marker, theflanking markers on each side generallyverified the recombination event. How-ever, in certain cases there appeared tobe a surplus of double recombinationevents. Treating such data as missing dataresolved inconsistencies in marker se-quence and shortened the linkage map inseveral regions. To avoid identifying falselinkages by such changes, we only re-ported linkages conforming to the strictconditions of an LOD score of at least 3.0and a recombination distance of no morethan 20 cM. Thus, in no case did thechanges identify new linkages or signifi-cantly alter the sequence of markers alonga linkage group. However, this treatmentdid shorten the total length of the applelinkage map by 50 cM-100 cM.We believe that the approach used toconstruct the linkage maps for apple isappropriate for a number of woody peren-nial crops for which relatively few parental

lines are used in breeding programs. RAPDmarkers permit an extensive map to begenerated quickly and relatively inexpen-sively, and such maps can be used to in-vestigate the genetic basis of importantmorphological or physiological variation.Tags for commercially important genessuch as those conferring disease resis-tance can be quickly identified, and be-cause relatively few parental sources areused, these tags can be widely applied inbreeding programs. We expect the use ofthe pseudotestcross and the double pseu-dotestcross design to greatly facilitatemapping in highly heterozygous plant spe-cies.ReferencesBeach SA, Booth NO, and Taylor OM, 1905. The appleof New York, 2 vols. NY Agric Exp Sin Rep 1903.Carlson JE, Tulsieram LK, Glaubitz JC, Luk VWK, Kauffeldt C, and Rutledge R, 1991. Segregation of randomamplified DNA markers in F. progeny of conifers. TheoAppl Genet 83:194-200.Cheliak WM, Morgan K, Dancik BP, Strobec KC, andYeh FC, 1984. Segregation of allozymes in megagametophytes of viable seed Irom a natural populationof jack pine. Pinus boanksiona Lamb. Theor Appl Gene69:145-151.Chevreau E an d Laurens F, 1987. The pattern of inheritance in apple (Malus x domestic Borkh.): furtheresults from leaf isozyme analysis. Theor Appl Gene75:90-95.Chevreau E, Lespinasse Y, and Gallet M, 1985. Inheritance of pollen enzymes and polyploid origin of applMalus x domestic Borkh.). Theor Appl Genet 71:268277.de Vicente MC and Tanksley SD, 1991. Genome-widreduction in recombination of backcross progeny derived from male versus female gametes in an interspecific cross of tomato. Theor Appl Genet 83:173-178Eckert RT, Joly R, and Neale DB, 1981. Genetics oisozyme variants and linkage relationships among allozyme loci in 35 eastern white pine clones. Can J FoRes 11:573-579.El-Kassaby YA, Sziklai 0, and Yeh FC . 1982. Linkagrelationships among 19 polymorphic allozyme loci icoastal Douglas-fir (Pseudotsuga menziesii var. 'menziesii'). Can J Genet Cytol 124:101-108.Grattapaglia D, Chaparro J, Wilcox P. McCord S.Werner D. Amerson H, McKeand S.Bridgwater F, WhetteR, O'Malley D, and Sederoff R, 1992. Mapping in woodplants with RAPD markers: application to breeding iforestry and horticulture. In: Applications of RAPDtechnology to plant breeding. Madison, Wisconsin: PlanScience Society of America; 37-40.Guries RP, Friedman ST, and Ledig FT. 1978. A megagametophyte analysis of genetic linkage in pitch pin(Pinus rigida Mill.). Heredity 40:309-314.Hagens DM, 1992. Genetic studies in Malus integratioof isozymes, DN A markers and morphological trait(MS thesis). Ithaca. Ne w York: Cornell University.Hamrick JL and Godt MJW, 1990. Allozyme diversiin plant species. In: Plant population genetics, breeding and genetic resources (Brown AHD, Clegg MT , Kahler AL. and Weir BS , eds). Sunderland, MassachusettsSinauer; 43-63.Jefferson RM, 1970. History, progeny and locations ocrabapples of documented authentic origin. NationaArboretum Contribution no. 2. Beltsville, MarylandARS/USDA.

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Molecular Marker Linkage Map for Apple