H O R TS C I E N C E 2 7 ( 2 ) : 1 6 0 - 1 6 3 . 1 9 9 2 .

Application of RFLP Analysis toGenetic Linkage Mapping in PeachesL. Eldredge, R. Ballard, W.V. Baird1, and A. AbbottDepartment of Biological Sciences, Clemson University, Clemson,SC 29634-1903

P. Morgens, A. Callahan, and R. ScorzaAppalachian Fruit Research Station, Agricultural Research Service, U. S.Department of Agriculture, Kearneysville, WV 25430

R . M o n e tINM Centres de Recherches de Bordeaux, 33883 Villenave D’OrnonCedex, France

Additional index words. Prunus persica, restriction fragment length polymorphism,genome mapping

Abstract. Peach [Prunus persica (L.) Batsch.] is considered the best genetically char-acterized species of the genus Prunus. We therefore used it as a model in our study ofthe genome organization in Prunus by means of restriction fragment length polymor-phisms (RPLPs). Initial results indicated that 60% of cloned DNA sequences examinedoccur at low copy number within the peach genome. After selecting and examiningthese sequences, polymorphisms sufficient for RPLP mapping were found. We deter-mined that »33% of our cDNA clones and 20% of our genomic clones detected RPLPsamong peach cultivars. Analysis of RPLP segregation in two families, both of whichsegregate for known morphological characters, revealed segregation in 12 RFLP mark-ers for one family and 16 for the other. Although we have not detected linkage betweenRFLP and morphological markers, preliminary analyses indicate possible linkage be-tween two RPLP markers.

Traditionally, the Rosaceae are divided intofour well-defined subfamilies. One subfam-ily, the Prunoideae, is characterized by spe-cies that produce drupes as fruits and containsseveral important fruit tree species, such aspeach/nectarine, within the genus Prunus.

Although Prunus is an important genus,little is known about the genome structureand organization of its members. To date,peach is considered the best genetically char-acterized species of the genus (Mowrey etal., 1990). Although no cytogenetic markershave been identified for peach, ≈35 mor-phological (Monet, 1989) and biochemical(Arulsekar et al., 1986) traits have been de-scribed, with two pairs of markers showinglinkage (Hesse, 1975; Monet et al., 1985;S.A. Mehlenbacher, personal communica-tion). In addition, heritability has been es-timated for another 20 traits (Monet, 1989).However, few monogenic (or qualitative)traits have been incorporated into individuallines to facilitate genetic studies. This un-derscores the extent to which the genetics ofpeach is still poorly understood.

Received for publication 3 June 1991. Acceptedfor publication 10 Sept. 1991. We thank J. Bel-thoff for editorial comments. This research wassupported in part by funds from the South CarolinaAgricultural Experiment Station Project 1310 andthe National Science Foundation - R11-8922165.The cost of publishing this paper was defrayed inpart by the payment of page charges. Under postalregulations, this paper therefore must be herebymarked advertisement solely to indicate this fact.1Department of Horticultural Sciences.

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Tremendous advances have been made inunderstanding the biology of simple andcomplex organisms through the developmentand use of genetic linkage maps (e.g., E.coli, Salmonella, yeast, Drosophila, Cae-norhabditis). Traditionally, the developmentof complete linkage maps requires the iden-tification of hundreds of single-gene muta-tions that govern easily scored phenotypictraits, and the availability of individuals froma segregating population such as an F2 orbackcross. The long generation time in treespecies severely limits the use of traditional

Fig. 1. Southern hybridization patterns of three tyby autoradiography: (A) a low-copy sequence proand (C) a highly repeated sequence probe.

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methods for genetic mapping. Molecular ge-netic approaches, however, enable one torapidly produce highly saturated maps fromexisting crosses. One such technique exam-ines the inheritance of RFLPs. Differencesin fragment lengths are consequences of her-itable changes in the DNA sequence struc-ture. Because RFLPs behave in a strictMendelian fashion and can be identified bymolecular hybridization when plants are veryyoung, these molecular markers are ideallysuited to genetic map construction. In higherplants, most mapping efforts have been re-stricted to herbaceous plants (see, for ex-ample, Bonierbale et al., 1988; Helentjariset al., 1986; Landry et al., 1987; Tanksleyet al., 1988).

We are extending the use of RFLP map-ping methods Prunus spp., beginning withthe development of a genetic map in peaches.In this paper, we present initial observationson the level of DNA fragment polymorphismin peach cultivars and demonstrate segrega-tion of RFLP markers in specific peach fam-ilies (parents and offspring).

Plant material. Prunus leaf samples col-lected from orchards at Clemson Univ. in-cluded: ‘Ruby Almond’, ‘Caramel Almond’,peach x [P. davidiana (Carrière) Franch], Sx R 185 peach x almond, NCA 10254 peachx almond, ‘Springcrest’, ‘Babygold #5’,‘O’Henry’, ‘Carolina Red’, ‘Cresthaven’,‘Stoneyhard’, ‘Redglobe’, ‘Ryan’s Son’, and‘Hakuto’. Leaves from 78 individuals in seg-regating populations (parents and offspring)from the Appalachian Fruit Research Stationin Kearneysville, W.Va. (hereafter referredto as the WV families) and 27 individualsfrom the INRA Centres de Recherches deBordeaux, Bordeaux, France (hereafter re-ferred to as the French family) were used forlinkage map construction.

DNA isolation and library construction.Genomic DNA was isolated from leaf tissueusing the procedure of Rogers and Bendich(1985), slightly modified to increase DNAyield. Modifications included grinding leaftissue with sand and increasing the amount

pes of random genomic inserts of peach detectedbe, (B) an interspersed-repeated sequence probe,

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Fig. 2. (A) Eco RI digested random genomic peach clones electrophoresed through a 0.8% agarosegel. (B) Southern hybridization pattern of random genomic clones shown in (A) detected by auto-radiography using nick-translated genomic peach DNA as a probe. Clones in lanes l-3 were shownby previous analysis to carry low-copy, interspersed-repeated, and highly repeated sequences, re-spectively, and were used as standards to judge the copy number of unknown clones. The arrowdenotes the position of the pUC8 plasmid used to internally standardize the loadings.

of leaf tissue and volumes of solutions, butkeeping the original ratios.

Probes for RFLP analyses were of twotypes: 1) randomly selected genomic clonesand 2) cDNA clones. Genomic libraries wereprepared from the peach cultivars Blake andBicentennial in the plasmid pUC8 (Messing,1983), by digesting total DNA with the re-striction enzyme Eco RI following the su-plier’s suggestions (Promega Biotech,Madison, Wis.). Plasmids carrying insertswere used to transform the bacterial host, aJM83 strain of E. coli (Maniatis et al., 1982).cDNA libraries were constructed in the vec-tor Lambda Zap (Stratagene, San Diego) fol-lowing Gubler and Hoffman (1983) andMorgens et al. (1990), except that double-stranded cDNAs were eluted over NENsorb(DuPont, Wilmington, Del.) columns, ac-cording to the manufacturer’s instructions,before ligation to the vector. Three cDNAlibraries were made from fruit RNA of thefollowing cultivars: 1) ‘Suncrest’ harvested30 days after bloom, 2) ‘Loring’ harvestedbetween 5 and 20 N of fruit firmness, and3) selection 612615 harvested at 140 daysafter bloom. These cDNA libraries werescreened by differential hybridization; the nine

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isolated clones represented genes whosetranscript accumulation was regulated duringfruit development. These clones were con-verted to pBluescript plasmids following themanufacturer’s instructions (Stratagene).

Genomic library screening. Any particu-lar clone in a genomic library may carry oneof three types of sequences: 1) low-copy, 2)interspersed-repeated, or 3) highly repeatedsequences. For genomic DNA probes to beuseful for RFLP analysis, they must carryDNA sequences that are present at uniqueloci within the genome. Therefore, to elim-inate clones with repeated sequences, a pre-screening method was used. Recombinantplasmids were digested with Eco RI to re-lease the inserts. Inserts were separated fromplasmids by electrophoresis through 0.8%agarose gels, blotted (Southern, 1975) ontoHybond-N membrane (Amersham, Arling-ton Heights, Ill.), and hybridized with nicktranslated, [α – 32P] dCTP (DuPont) la-belled, genomic peach DNA (Maniatis et al.,1982) at 65C. DNA clones previously de-termined to carry low-copy, interspersed-re-peated, and highly repeated sequence DNA(as described below) were included on eachgel to serve as comparative standards for es-

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Detection of RFLPs. Genomic DNA fromvarious cultivars was digested with severalrestriction enzymes, including sir-base (HindIII, Eco R1) and four-base cutters (Sau 3A,Hae III), and samples of each were electro-phoresed on a 0.8% agarose gel for ≈16 hat 45 V. Phage λ DNA digested with HindIII was included as a molecular weight marker.Gels were treated and blotted onto nylonmembranes (Southern, 1975) and mem-branes were hybridized with either pre-screened genomic DNA or cDNA probes. Toprepare the probes, recombinant plasmidswere purified and digested to release the in-serts, which were then isolated by electroe-lution from agarose gels (Maniatis et al.,1982). Genomic probes were either nicktranslated (Maniatis et al., 1982) or randomprimed (Feinberg and Vogelstein, 1983) toincorporate the radioactive signal. IsolatedcDNA inserts were random primed using akit from BRL (Gaithersburg, Md.). Filterswere hybridized as previously described.Following autoradiography, filters werestripped of the radioactive probe for reuse byshaking gently in a 55% formamide, 2xSSPE (0.15 M NaCl, 0.01 M sodium phos-phate, 0.001 M EDTA, pH 7.4), 1% sodiumdodecyl sulfate (SDS) solution at 65C for 45to 90 min followed by a 1-min rinse in 0.1 ×SSC (0.15 M NaCl, 0.015 M trisodium cit-rate, pH 7.0) and 0.1% SDS.

Our long-term objective is to obtain a high-resolution RFLP map of the peach genome.To begin preliminary work toward this goal,a shotgun plasmid library of restriction-di-gested genomic peach DNA and a cDNAlibrary from developing fruit were preparedto obtain a collection of clones to be used asprobes in Southern hybridization analyses.Initially, clones were randomly chosen fromthe genomic plasmid library, and their in-serts were used as probes to determine theproportion of clones in the library that hadinserts of low-copy sequences. Clones car-rying low-copy, interspersed-repeated, andhighly repeated sequences were identified(Fig. 1). From these initial screenings, weestimated that 60% of 32 clones carried low-copy sequences. Because 40% of clones car-ried sequences of little use in our analyses(i.e., interspersed-repeated and highly re-peated sequences) a prescreening method wasused to eliminate clones carrying repeatedsequences. After prescreening, only clones

timating copy number of each insert beingscreened. Autoradiography was carried outby placing filters in cassettes equipped withone Cronex Lightning Plus intensifying screen(DuPont). Kodak X-OMAT XAR-5 X-rayfilm was placed on the hybridized filters andcassettes were placed at –80C for 2- to 10-day exposures (depending on the strength ofthe hybridization signal). Following autora-diography, low-copy sequences were de-fined as those that showed little or no signal,interspersed-repeated sequences were thosethat gave faint but distinct signals, and highlyrepeated sequences showed very strong sig-nals. Only clones judged to carry low-copysequences were further analyzed as potentialRFLP probes.

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Fig. 3. (A, B) Southern hybridization patterns of two cDNA probes of peach detected by autoradiog-raphy. Genomic DNA of each was digested with Hind III and probed with either pch 103 (A) or pch205 (B). Lane 1, ‘White Glory’; lane 2, RRL-2N, lane 3, 174-RL; lane 4, ‘Marsun’; lane 5, ‘DavieII Dwarf’; lane 6, ‘NC Pillar’; lane 7, ‘Honey Gold’; lane 8, ‘Redskin’; lane 9, ‘NJ Pillar’; lane 10,77119; lane 11, ‘Armking’; lane 12, P19-11; lane 13, 77017; lane 14, ‘Mission Almond’ [Prunusdulcis (Mill.) D.A. Webb]; and lane 15, P. davidiana.

judged to be low-copy were further analyzedas potential RFLP probes (Fig. 2).

Previous isoenzyme studies suggest thatthe genetic base of peach is fairly narrow andthat enzyme polymorphisms are quite rare(Durham et al., 1987; Mowrey et al., 1990). Therefore, initial efforts to generate an RFLPmap were directed at assessing the extent ofDNA polymorphism in peach cultivars. Fourof 23 probes detected polymorphisms in thevarious cultivars, suggesting that the degreeof polymorphism was sufficient for RFLPmapping. This degree of polymorphism wasnot as high as that detected in interspecificcomparisons, where we estimated the levelof polymorphism to be >50% (i.e., 13 of25 using Hind III). However, to avoid prob-lems of map compression evident in inter-specific crosses (Paterson et al., 1990), weworked only with crosses within P. persica.

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Because cDNA clones represent se-quences that are spatially and temporallyregulated in tissues from which template RNAwas isolated, they are ideally suited for in-vestigating levels of polymorphism in genesor gene-flanking regions. We isolated ninecDNA clones that were complementary togenes differentially regulated during peachfruit development (Callahan et al., 1991).When used to screen for RFLPs among avariety of peach and almond cultivars and apeach x almond hybrid, three of the nineclones detected RFLPs among the peach cul-tivars. Only one clone did not detect an RFLPbetween the peach and almond cultivars. Ofthe nine clones, one represented a small re-petitive family of related genes, one rep-resented a small family of genes (perhaps10), and the remaining seven represented oneto three related genes each. Our analysis in-

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dicated that ≈33% of cDNA clones may de-tect RFLPs among commercial peach cultivars(Fig. 3).

Because one of the goals of our researchwas to link RFLP characters to agriculturallyimportant morphological traits, progeny frompeach crosses that exhibited segregation forboth morphological characters and RFLPmarkers were necessary for map construc-tion. Families that segregate for morpholog-ical traits need to be analyzed to determinethe extent of segregation of RFLPs withinthese individuals.

Individuals resulting from the ‘Jalhousia’x ‘Summergrand cross (i.e., the Frenchfamily) showed segregation for three mor-phological characters: peach shape, pollenfertility, and peach/nectarine. To date, wehave discovered probes that show segrega-tion for 12 RPLP loci in the French family(Fig. 4A). Here, parent 1 (lane 1) is homo-zygous for the locus; it has two copies of thehigh molecular weight band. Parent 2 (lane2) is a heterozygote, having one copy of thehigh molecular weight band and one copy ofa pair of smaller bands. These smaller bandsmay have resulted from the addition of aHind III restriction site to the high molecularweight band. The progeny (lanes 3-13) showthe predicted 1:1 segregation for the allelesof this locus. To date, we have not detectedlinkage of a morphological character to anRFLP. Preliminary analyses, however, in-dicate possible linkage between two RFLPs.

The WV families consist of two parents,four F1 offspring, and the F2 progeny fromthe self-fertilized F1 individuals. Individualswithin this family show segregation for thegenetic loci that influence tree shape and size.We have discovered probes that show seg-regation for 16 RFLP loci in this family.Figure 4B illustrates the segregation of anRFLP locus in one WV family, where theparents (lanes 1 and 2) are each homozygousat the RFLP locus, but each for a differentallele. As expected, the F1 individual (lane3) is heterozygous for the alleles at this lo-cus, inheriting one copy from each parent,and the F2 progeny (lanes 4-14) show theexpected F2 segregation classes for the twoalleles. We detected seven probes that showedpolymorphism in both the French and WVfamilies, allowing us to combine the map-ping information from the data sets of theseunrelated individuals. We used MAP-MAKER (Lander et al., 1987) to statisticallyevaluate our data for linkage.

In conclusion, we demonstrated that peachdisplays sufficient DNA polymorphism toallow the construction of a molecular geneticmap. This map will serve as a foundation tostudy genome organization and evolution inother Prunus spp.

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Fig. 4. (A, B) Southern hybridization patterns of polymorphic probes of peach detected by autora-diography. Genomic DNA was digested with Hind III and probed with either B2A10 (A) or B4F12(B). (A) French family: lane 1, ‘Jalhousia’; lane 2, ‘Summergrand’; and lanes 3–15, F 1 plants. (B)WV family: lane 1, ‘NJ Pillar’; lane 2, 77119; lane 3, F 1 plant, and lanes 4–15, F2 plants producedby self-pollinating the F1.

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