Genetic Analysis of Restriction Fragment Length Polymorphisms in Vitis M.-C. Mauro, M. Strefeler, N. F. Weeden, and B. I. Reisch From the Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456. Dr. Mauro is presently at Moet-Chandon, Epernay, France, and Dr. Strefeler is presently at the Department of Horticultural Science, University of Minnesota, St. Paul. This work was sup- ported in part by a grant from the Cornell Biotech- nology Program, which is sponsored by the New York State Science and Technology Foundation, a consor- tium of industries, the U.S. Army Research Office, and the National Science Foundation. The authors thank M. Hemmat for technical assistance and M. P. Pritts and two anonymous reviewers for many helpful com- ments. Address reprint requests to N. F. Weeden at the address above. Journal of Heredity 1992;83:18-21; 0022-1503/92/$4.00 The parents and progeny from two crosses (Cayuga White x Aurore and NY62.136.2 x Yates) were examined for the presence of DNA restriction fragment length poly- morphisms (RFLPs). Seventeen independent DNA sequences were used in the anal- ysis, 15 obtained from a grape Pst\ genomic library and two heterologous probes obtained from other laboratories. Most of the low copy cloned sequences hybridized to more than two restriction fragments, possibly reflecting the polyploid nature of the Vitis genome. Nine of the probes detected RFLPs between parents. Analysis of the progenies (F, generation) revealed segregation for nine distinct polymorphisms generated by seven of the probes. Thus, a relatively high level of polymorphism among parents, as well as heterozygosity within each parent, was evident. Most RFLPs gave segregation ratios close to the 1:1 ratio predicted for a locus hetero- zygous in one parent. However four differences between parental phenotypes did not segregate in the progeny, and in three instances fragments present in both parents segregated in the progeny. These peculiar results may be explained by accounting for heterozygosity or homozygosity, respectively, for the DNA segment that generates the polymorphism. We conclude that RFLP studies can be performed on the first filial generation in woody perennials such as Vitis that have a relatively high level of heterozygosity in the genome. Despite the fact that grape ( Vitis spp.) is the oldest and most widely grown fruit crop in the world, few simply inherited char- acters have been identified in this genus. The crop has not been amenable to genetic analysis because of its relatively long generation time (2 to 5 years), its highly heterozygous nature and sensitivity to in- breeding depression, and a dearth of well- defined genetic markers. Genetic analyses of polymorphisms among 48 morpholog- ical traits have revealed that they are con- trolled by distinct loci (De Lattin 1957). However, the patterns of inheritance of many of these traits have not been well characterized, and the availability of clones with mutant characters is limited. Genetic studies of many annual crop species have been augmented by the use of molecular markers such as allozymes or DNA restriction fragment length poly- morphisms (RFLPs), and such markers will be important tools for plant breeders and geneticists. The widespread use of protein electrophoresis in woody crops has oc- curred only recently. A number of woody taxa, including several conifers (El-Kas- saby et al. 1982; Harry 1986; Millar 1984), Eucalyptus(Moran and Bell 1983), and Ma- /us(Chevreauetal. 1985; Weeden and Lamb 1987) display relatively high levels of allo- zyme polymorphism, whereas others pos- sess only a few disernable allozyme vari- ants. RFLP studies can result in the placement of thousands of genetic mark- ers in each species, many more than are possible with allozyme analysis. However, RFLP technology currently requires a con- siderable investment of time and money. Thus, it would be advantageous to be able to predict which woody taxa will be most conducive to this approach. Although a comprehensive study has yet to be made, there appears to be a positive correlation in herbaceous species between the level of allozyme variability present in a taxon and the ease with which RFLPs can be found in that taxon (Chao et al. 1989). This correlation may merely reflect an increase of both types of polymorphisms with ge- netic diversity. Nevertheless, because allozyme data are often available for spe- cies in which RFLP studies have yet to be performed, the correlation could prove to be very useful to investigators who are ex- panding RFLP technology to new taxa. The purposes of this study were to determine (1) if the relatively high level of allozyme 18
Transcript
Genetic Analysis of Restriction FragmentLength Polymorphisms in VitisM.-C. Mauro, M. Strefeler, N. F. Weeden, and B. I. Reisch
From the Department of Horticultural Sciences, NewYork State Agricultural Experiment Station, CornellUniversity, Geneva, NY 14456. Dr. Mauro is presentlyat Moet-Chandon, Epernay, France, and Dr. Strefeleris presently at the Department of Horticultural Science,University of Minnesota, St. Paul. This work was sup-ported in part by a grant from the Cornell Biotech-nology Program, which is sponsored by the New YorkState Science and Technology Foundation, a consor-tium of industries, the U.S. Army Research Office, andthe National Science Foundation. The authors thankM. Hemmat for technical assistance and M. P. Prittsand two anonymous reviewers for many helpful com-ments. Address reprint requests to N. F. Weeden atthe address above.
Journal of Heredity 1992;83:18-21; 0022-1503/92/$4.00
The parents and progeny from two crosses (Cayuga White x Aurore and NY62.136.2x Yates) were examined for the presence of DNA restriction fragment length poly-morphisms (RFLPs). Seventeen independent DNA sequences were used in the anal-ysis, 15 obtained from a grape Pst\ genomic library and two heterologous probesobtained from other laboratories. Most of the low copy cloned sequences hybridizedto more than two restriction fragments, possibly reflecting the polyploid nature ofthe Vitis genome. Nine of the probes detected RFLPs between parents. Analysis ofthe progenies (F, generation) revealed segregation for nine distinct polymorphismsgenerated by seven of the probes. Thus, a relatively high level of polymorphismamong parents, as well as heterozygosity within each parent, was evident. MostRFLPs gave segregation ratios close to the 1:1 ratio predicted for a locus hetero-zygous in one parent. However four differences between parental phenotypes didnot segregate in the progeny, and in three instances fragments present in bothparents segregated in the progeny. These peculiar results may be explained byaccounting for heterozygosity or homozygosity, respectively, for the DNA segmentthat generates the polymorphism. We conclude that RFLP studies can be performedon the first filial generation in woody perennials such as Vitis that have a relativelyhigh level of heterozygosity in the genome.
Despite the fact that grape ( Vitis spp.) isthe oldest and most widely grown fruit cropin the world, few simply inherited char-acters have been identified in this genus.The crop has not been amenable to geneticanalysis because of its relatively longgeneration time (2 to 5 years), its highlyheterozygous nature and sensitivity to in-breeding depression, and a dearth of well-defined genetic markers. Genetic analysesof polymorphisms among 48 morpholog-ical traits have revealed that they are con-trolled by distinct loci (De Lattin 1957).However, the patterns of inheritance ofmany of these traits have not been wellcharacterized, and the availability of cloneswith mutant characters is limited.
Genetic studies of many annual cropspecies have been augmented by the useof molecular markers such as allozymesor DNA restriction fragment length poly-morphisms (RFLPs), and such markers willbe important tools for plant breeders andgeneticists. The widespread use of proteinelectrophoresis in woody crops has oc-curred only recently. A number of woodytaxa, including several conifers (El-Kas-saby et al. 1982; Harry 1986; Millar 1984),Eucalyptus(Moran and Bell 1983), and Ma-
/us(Chevreauetal. 1985; Weeden and Lamb1987) display relatively high levels of allo-zyme polymorphism, whereas others pos-sess only a few disernable allozyme vari-ants. RFLP studies can result in theplacement of thousands of genetic mark-ers in each species, many more than arepossible with allozyme analysis. However,RFLP technology currently requires a con-siderable investment of time and money.Thus, it would be advantageous to be ableto predict which woody taxa will be mostconducive to this approach. Although acomprehensive study has yet to be made,there appears to be a positive correlationin herbaceous species between the levelof allozyme variability present in a taxonand the ease with which RFLPs can befound in that taxon (Chao et al. 1989). Thiscorrelation may merely reflect an increaseof both types of polymorphisms with ge-netic diversity. Nevertheless, becauseallozyme data are often available for spe-cies in which RFLP studies have yet to beperformed, the correlation could prove tobe very useful to investigators who are ex-panding RFLP technology to new taxa. Thepurposes of this study were to determine(1) if the relatively high level of allozyme
18
polymorphism found in Vitis is accompa-nied by similarly abundant RFLPs and (2)if these RFLPs can be genetically analyzedand mapped using Fi plants, an approachthat has been successfully applied to iso-zyme loci in several woody perennials.
Materials and Methods
The parents and unselected progeny of twocrosses (81.316: Cayuga White x Auroreand 78.839: NY62.136.2 x Yates) weregrown in the vineyards of the New YorkState Agricultural Experiment Station, Ge-neva, New York. The parents of thesecrosses were complex interspecific hy-brids. The progeny of the first cross waspreviously surveyed for allozyme poly-morphisms (Weeden et al. 1988).
We prepared high molecular weight DNAfrom young leaf tissue of the parents andprogeny according to the method of Doyleand Doyle (1987). Approximately 7 figsamples of this DNA were digested over-night using six restriction endonucleases(£coRI, £coRV, BamHl, HindlU Bgl\, andXbaY) according to the manufacturer's in-structions. After separation by electropho-resis on 1% agarose gels, the DNA frag-ments were transferred to nylonmembranes (GeneScreen Plus, Dupont,Wilmington, Delaware) by using a modi-fication of the techniques of Southern(1975) in which the DNA was transferredto the membrane under alkaline condi-tions (Reed and Mann 1985).
We developed a partial genomic libraryfrom high molecular weight DNA isolatedfrom young leaves of V. vinifera cv. Caber-net Sauvignon. The DNA was digested with0.12 units of the restriction endonucleasePst\ per 1.0 ng DNA for 1 h at 37°C. Ap-proximately 40.0 ng of this restricted DNAwere ligated into 1.0 tig of Pst\ cut pBR322(New England Biolabs, Beverly, Massa-chusetts). We incubated the restricted DNAand Pst\ cut pBR322 for 4 h at room tem-perature in 200 mM Tris (pH 6.0), 100 mMdithiothreitol, 6 mM ATP, and 1 id of T4ligase. The ligated DNA was used to trans-form E. coli DH5a competent cells accord-ing to the manufacturer's instructions (LifeTechnologies, Inc., Gaithersburg, Mary-land). Transformed cells were stored at-70°C. We screened these clones for thepresence of repetitive DNA inserts by ex-tracting the recombinant plasmids via arapid plasmid isolation procedure (Wil-imzig 1985), blotting 10 ng of plasmid DNAon a nylon membrane, and hybridizingthem against 32P-labeled chloroplast DNAof Solarium hyporhodium and 32P-labeled
total cellular DNA from V. vinifera. Plas-mids containing inserts failing to hybrid-ize to the cpDNA and displaying weak hy-bridization to the total cellular DNA wereused as probes for the parental screening.We also used two heterologous clones—the soybean actin gene (pSAc3, Shah et al.1982) and a carrot genomic clone (A-56,provided by Dr. Phil Simon, USDA-ARS,Madison, Wisconsin)—to identify RFLPs.
Probe DNA was nick translated follow-ing the protocols of Maniatis et al. (1982)except that four units of DNase I/Poly-merase I (Life Technologies, Inc., Gaith-ersburg, Maryland) were added to the 30-fi\ reaction mixture instead of adding eachenzyme separately. We incubated the nicktranslation mixture for 1-2 h at 14°C, afterwhich we separated the labeled probesfrom unincorporated nucleotides by spuncolumn chromatography on a SephadexG-50 1 cc column following the method ofManiatis et al. (1982). Before hybridiza-tion, the labeled probes were denaturedin solution with IN NaOH at room tem-perature for 10 min.
We prehybridized the blots for 4-6 h at65°C in approximately 10 ml of buffer per100 cm2 of blot. The hybridization bufferconsisted of 750 mM NaCl, 125 mM citricacid (trisodium salt, dihydrate), 2.5 mMEDTA, 50 mM NaPO4, 5 x Denhardt's, 0.4mg/ml calf thymus DNA, 5.0% dextran sul-fate, and 0.6% SDS. Hybridization was car-ried out in the same buffer for 16-18 h at65°C after adding denatured radiolabeledprobe. After hybridization, the blots werewashed two to three times in an excess of2 x SSC + 0.1% SDS for 20 min at 65°C. Weperformed more stringent washes (1 x SSCand 0.1 x SSC) on several occasions in at-tempts to simplify the pattern generatedon autoradiograms. Autoradiography wascarried out at -70°C. Segregation and link-age analyses were carried out using theLINKAGE-1 program of Suiter et al. (1983).Initially, we scored each segregating re-striction fragment as being present or ab-sent. Identification of allelic fragments oc-casionally permitted the heterozygousphenotype to be recognized. If both par-ents were heterozygous, the data werereanalyzed using an expected 3:1 or 1:2:1segregation ratio. The allozyme segrega-tion data used in the linkage analyses werepreviously reported (Weeden et al. 1988).
Results and Discussion
High molecular weight DNA was obtainedin good yield from young leaf tissue. Bestresults were obtained on rapidly expand-
ing leaves one to two nodes from the shoottip. Older leaves, especially those fully ex-panded, tended to give loweryields of DNA,and this DNA could not be cut to comple-tion with the restriction endonucleases.
A small genomic library was generatedfrom Cabernet Sauvignon. Approximately200 recombinant plasmids were isolated;however, only 15 of these met the condi-tions of the screen and generated rela-tively few clear bands on autoradiograms.Of the 17 independent probes screened(15 grape genomic and two heterologous),nine displayed polymorphism between theparents of one or both crosses (Table 1).Most of the polymorphisms could be de-tected using £coRI-cut DNA. Other en-zymes also could have been used to scorefor RFLPs (for example, pGG224 showedpolymorphism for HindUl and XbaY); how-ever, the number of blots required for theanalysis was minimized by using £coRIpolymorphisms when they were available.Fifteen of the 17 probes generated auto-radiograms with more than two fragmentshybridizing to the probe DNA. In thosecases tested (pGG65, pGG224, pGG319),the observed pattern was not simplifiedsignificantly when higher stringencywashes were performed.
Seven of the nine probes displaying in-terparent polymorphism also showed seg-regation in the progenies (Table 2). Fiveof these probes (pGG75, pGG243, pGG254,pGG276, and A56) visualized just one poly-morphic fragment, segregating as eitherpresent or absent in the phenotype. Thesegregation of each of these approximatedthe expected 1:1 ratio for a single hetero-zygous locus (Table 2), although for pGG75the deviation from 1:1 was significant at P= .05.
The probe pGG224 revealed two banddifferences between each pair of parents,but only one of these polymorphisms seg-regated in each progeny (Table 2). Thetwo segregating polymorphisms gave a 1:1phenotypic ratio in the progenies, indi-cating that the parent possessing the re-striction fragment was heterozygous for therestriction site(s) generating that frag-ment. The two nonsegregating polymor-phisms were uniformly present in the re-spective progeny, suggesting that theparent with the restriction fragment washomozygous for the appropriate restric-tion sites. The two probes, pGG238 andpSAc3, also gave results similar to the non-segregating polymorphisms of pGG224.Band intensity, as measured visually, wasnot particularly helpful for discriminatingbetween homozygous and heterozygous
Mauro et al • RFLPs in Vitis 19
Table 1. Clones evaluated, number of restrictionfragments resolved by each clone, andendonucleases most practical for resolvingpolymorphisms
°pGG26, pGG57, pGG65, pGG73, pGGllO, pGGll l ,pGG219, and pGG269 generated autoradiograms with1 -7 bands, all of which were invariant among the parentsand the progenies examined.
Table 2. Single-locus segregations and chi square analyses for RFLPs in segregating grapevine F,populations
Figure 1. fcoRl restriction length phenotypes generated by pGG319. The upper portion of the figure shows theAurore phenotype in the left lane and individual progeny from the Cayuga White x Aurore cross in the remaining10 lanes. The lower section diagrams all phenotypes. Designations are PI = Cayuga White or NY62.136.2; P2 =Aurore or Yates; A, B, C, and D = four phenotypes observed in progenies; M = ///ndlll cut lambda DNA.
genotypes, and we were unable to predictfrom the band intensity in the parentalpattern whether that band would segre-gate in the progeny.
Plasmid pGG319 generated a relativelycomplicated set of phenotypes in whichfour restriction fragments segregated (Fig-ure 1). The same two parental phenotypeswere present in both crosses, those forCayuga White and NY62.136.2 being iden-tical, as were those for Aurore and Yates.Except for minor differences in the inten-sity of certain bands, the only differencebetween the two phenotypes was that Ca-yuga White and NY62.136.2 possessed a10-kb fragment, whereas the other phe-notype lacked such a band (Figure 1). Allfour fragments displayed segregation inboth progenies, yet only four phenotypeswere observed. We interpreted these re-sults to indicate that each parent must beheterozygous and that at least three allelesmust be present in each cross. Accordingto our model, Cayuga White and NY62.136.2possess one "allele" (DNA sequence) thatgenerates a 13-kb and a faint 7.5-kb frag-ment and a second allele that generates10- and 6-kb fragments. In contrast, Auroreand Yates possess an allele that generatesonly a 6-kb fragment as well as an allelethat produces the 13-kb and faint 7.5-kbfragments. The model predicts that the 13-kb, 7.5-kb, and 6-kb fragments should eachshow 3:1 segregation ratios, and the 10-kbfragment should segregate in a 1:1 ratio.In addition, the four phenotypes distin-guishable in the progenies should segre-gate in a 1:1:1:1 ratio. All the predictionsof the model were fulfilled (Tables 2 and3)-
Joint segregation analysis of the RFLPsand the isozyme loci described previously
20 The Journal of Heredity 1992:83(1)
Table 3. Segregation analysis of pGG319 phenotypes
Cross
81.31678.839
N
5334
A
175
B
1811
C
00 00
D
1010
Expectedratio
1:1:1:11:1:1:1
X2
5.642.47
P
.1-.2
.3-.5
"Phenotypic designations are those as given in Figure 1, which represent the four patterns observed onautoradiograms.
(Weeden et al. 1988) failed to identify anynew linkage groups for grape. This failureto find additional linkages is not particu-larly surprising when one considers that,in this type of analysis, the segregating locimust be heterozygous in the same parentfor linkage to be observed. The nine dis-tinct (i.e., assorting independently or indifferent F, progenies) segregating phe-notypes listed in Table 2 were distributedamong four parents. Thus the number ofactual comparisons available for linkageanalysis is much lower than the total num-ber of loci investigated. However, this studyclearly demonstrates that RFLPs are prev-alent in Vitis. Nine segregating loci couldbe scored after screening 17 low copy nu-clear sequences.
The relatively high number of fragmentshybridizing to most probes may indicatethat most low copy DNA fragments arepresent as two or more closely related DNAsequences in the grape genome. Olmo
(1976) indicated that grape probably is ofpolyploid origin. The multiplicity of close-ly related sequences in the genome mayreflect its polyploid nature. Continuedanalysis of the Cayuga White x Auroreandother suitable progenies should permit thedevelopment of a complete linkage mapfor grape, as well as a detailed geneticcharacterization of this species.
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