Linkage between Pgm-p, the locus specifying the plastid specific phosphoglucomutase, and Mo, the locus controlling resistance to bean yellow mosaic virus (BYMV), was investigated in the garden pea, Pisum sativum L. Both genes are known to be on chromosome 2 and exhibit linkage with two morphological marker loci, K and Wb. Our results indicate that the gene order is: Wb—K—Pgm-p—Mo. A map distance of approximately two recombinant units was determined for the Pgm-p—Mo linkage, suggesting that the enzyme locus can be used as a genetic marker for resistance to BYM

Marx, G. A.

Provvidenti, R.

Weeden, N. F.

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NotesThe Journal of Heredity 75:411-412. 1984.An isozyme marker forresistance to bean yellowmosaic virus in Pisum sativumN. F. Weeden, R. Prowldentl, andG. A. MarxABSTRACT Linkage between Pgm-p, the locusspecifying the plastk) specific phosphoglucomu-tase, and Mo, the locus controlling resistance tobean yellow mosaic virus (BYMV), was investi-gated in the garden pea, Plsum sativum L. Bothgenes are known to be on chromosome 2 andexhibit linkage with two morphological markerloci, /Cand Wb. Our results indicate that the geneorder is: Wb—K—Pgm-p—Mo. A map distanceof approximately two recombinant units was de-termined for the Pgm-p—Mo linkage, suggestingthat the enzyme locus can be used as a geneticmarker for resistance to BYMV.THE POTENTIAL use of polymorphic enzymeloci as genetic markers has been recognized eversince the simple genetic basis of allozymic vari-ants was demonstrated2'8. The technique was firstapplied in the tomato after Rick and Fobes5 de-scribed the tight linkage between an acid phos-phatase locus and the gene bestowing nematoderesistance. Few other examples of a close linkagebetween a polymorphic enzyme locus and acommercially important trait are available.However, polymorphic isozyme loci have beenused to analyze the genetic basis of a number ofquantitative characters9'10. The partial isozymelinkage map we described for Pisum sativumL.12 should provide markers for genes controllingseveral commercially important characters. Weare especially interested in those genes affectingdisease resistance, for such characters are oftenrevealed only after elaborate tests.Yen and Fry13 demonstrated that certain peacultivars carried resistance to bean yellow mosaicvirus (BYMV). Genetic tests showed that theresistance was conferred by a recessive allele atMo, a locus on chromosome 2 about five mapunits from a well-characterized flower mutant,The authors are affiliated with the Department ofHorticultural Sciences (N.F.W. and G.A.M.), and theDepartment of Plant Pathology (R.P.), New YorkAgricultural Experiment Station, Cornell University,Geneva, NY 14456.© 1984, American Genetic Association.A:3. Further studies on the mo gene have dem-onstrated that it, or a tightly linked gene, confersresistance to watermelon mosaic virus 27. Therelatively close linkage between k and mo haspermitted the former to be used as a marker formo in segregating progeny. However, severaldisadvantages are associated with using A: as amarker. The mutant phenotype is fully recessive,permitting the differentiation of only two prog-eny classes, and the phenotype is expressed in theflower, thus delaying the screening operationuntil the plants bloom. We report here a linkagebetween mo and the locus Pgm-p, which codesfor the plastid specific form of phosphogluco-mutase (PGM-2, EC 2.7.5.1). The distinctiveproperties of isozyme loci such as codominantand early expression of alleles and freedom frompleiotropic or epistatic interactions would indi-cate that Pgm-p may be superior to A: as amarker for mo.Materials and MethodsFive seed, representing the F3 generation fromB77-254 X A78-237 and the reciprocal cross,were obtained from each of 85 plants from the F2population A, described by Weeden and Marx12.The line B77-254 is resistant to BYMV (mo/mo), homozygous for the fast allele of Pgm-pand homozygous dominant at K and Wb. Theinbred line A78-237 is susceptible to BYMV,homozygous slow at Pgm-p, and exhibited therecessive phenotypes for k and wb. Seed weregerminated in an artificial soil mix and grown ina greenhouse at 20-23 °C. Seedlings were me-chanically inoculated with a type strain ofBYMV6 on the first two fully expanded leaves.To assure infection in all susceptible genotypes,plants were subsequently reinoculated on thethird and fourth leaves. Inoculum was preparedby triturating leaves of BYMV-infected peaplants with 0.05 M phosphate buffer (K+), pH7.0. Control plants from each parental line as wellas two check lines, 'Bonneville' (R) and 'Ranger'(S), were treated similarly to the F3 plants.The genotype at Pgm-p was determined onyoung leaf tissue by starch gel electrophoresis ona pH 8.1 tris-citrate/lithium borate gel systemas described previously1 '.Epicuticular wax development on both sur-faces of the stipules, the lower surface of theleaflets, and the pods is markedly reduced inwb/wb plants; stem wax also is somewhat re-duced1. The phenotype of mutant plants can bescored in the seedling stage. In k/k plants thewings (alae) of the flowers are smaller thannormal and tightly appressed to the keel1. The kphenotype was determined as soon as a plantbegan to flower.Linkage estimates were calculated as de-scribed previously12.ResultsAlthough all seed selected from the F2 plantsappeared normal, eight sets of five did not ger-minate and another seven sets produced only oneor two mature plants. Of the remaining 70 sets,66 exhibited 100 percent germination, three gavefour seedlings and one produced three seedlings.A total of 355 F3 plants were scored for thosecharacters (wb, mo, and PGM) that were ex-pressed in young plants. Homozygous (Mo/Mo)and heterozygous (Mo/mo) plants susceptible toBYMV responded to viral infection with a per-sistent yellow mosaic and stunting. Plants pos-sessing mo/mo were highly resistant and werefree of local and systemic infection. A number ofsusceptible plants died before flowering so thatthe data for k were not as complete. Controlplants responded as anticipated, with all fiveB77-254 plants showing a resistant phenotypeand all A78-237 plants being susceptible.Each character scored gave phenotypic ratiosin the F2 and F3 generations close to those ex-pected for two alleles segregating at a single locus(Table I). The ratios observed in the F3 wereslightly biased in favor of the recessive phenotypebecause only sets exhibiting both phenotypeswere included in the calculation. For instance, afamily consisting of three recessive and twodominant phenotypes would be included in thesummation, but a progeny in which all five plantsexhibited the dominant phenotype would bescored as not segregating even though there wasa significant probability (23.7 percent) that suchsets were derived from a heterozygous parent andwould show segregation if a larger sample hadbeen taken.Recombination frequencies between Mo andeach of the three other loci were calculated usingthe genotypes of the F2 plants as determined withthe aid of F3 progeny tests (Table II). This pro-cedure permitted the identification of most F2individuals heterozygous at Mo, Wb, or K, asdescribed above. The Telative recombinationTable L Segregation of alleles at Mo, K, Wb, and Pgtn-p for both the F2 and Fj generationsNo. plants with designated phenotype*F2 plants F) plantsLociMoKWbPgm-p-17241922H44+54615517X20.040.470.020.35-48344136H84+90769250X2'7.85**2.052.412.33* Designations: recessive phenotype or homozygous slow :fast = +* Bias not corrected for in x2 calculations• • P <0.01, but see above footnote and discussion in text—, heterozygous = H; dominant or homozygousNotes September / October 1984 411Table II. Joint segregation data in the Fi population for pairwise comparisons of the loci Mo,Pgm-p, K, and WbLociMo: Pgm-pMo.KMo.WbPgm-p:KPgm-p:WbN6560696771-/-0012015No." /H22414plants with designated phenotype*- / +15151600H / -00114H/H2916162520H/+1812513+ / -19181500+/H01403+/+0001512Recomb.fract.2918518SE1.242.753.681.933.63* Designations as described in Table Ifrequencies between loci indicated that the genesequence is: Wb—K—Pgm-p—Mo. Pgm-p ex-hibited tighter linkage with Mo (2 percent re-combinants) than with K (5 percent recombi-nants).The linkage Wb—K—Mo is consistent withprevious findings4; however, in order to determineif our linkage results were significantly biased bytreating F2 plants with nonsegregating progenyas homozygous at the particular locus (e.g., Wb,K, or Mo), two additional linkage calculationswere performed. These latter calculations werebased only on observable phenotypes within ageneration. The first considered only data ob-tained from the F2 population, treating the mo,wb, and k phenotypes as strictly dominant andrecessive. The second linkage calculation wasbased on F3 generation data alone and was lim-ited to those progenies segregating both at Moand at least one of the other linked loci. Bothcalculations (data not shown) gave a gene orderidentical to that obtained from results presentedin Table II. Furthermore, in both cases Pgm-pwas more tightly linked to Mo (4 percent re-combinants in the F2 and 1 percent recombinantsin the F3) than to K. This consistency implies thatour original linkage results accurately reflect thegene order and approximate recombinationfrequencies among the four loci.DiscussionThe demonstration that Pgm-p was locatedapproximately five map units from K on the sideopposite Wb12 led to the suggestion that thisisozyme locus could serve as an excellent markerfor mo, the locus controlling resistance toBYM V. The data presented in this study confirmthis possibility and indicate that the distancebetween Pgm-p and Mo is about two map units.Considering all 355 F3 plants analyzed for bothcharacters, the mo phenotype would have beenincorrectly predicted by the PGM-2 phenotypein only 11 instances, of which nine were progenyof the three recombinant F2 plants. Pgm-pproved to be superior to k for following the in-heritance of alleles at Mo not only because of thetighter linkage between Pgm-p and Mo but alsobecause of the ability to distinguish all threeclasses of progeny (both homozygotes and theheterozygote) and obtain these results on youngseedlings, even before it was possible to determinethe mo phenotype of the plants. Pgm-p alsoshould be useful as a marker for resistance towatermelon mosaic virus 2 whether or not mo ora tightly linked gene is responsible for this resist-ance.Infection of the leaf tissue by the virus did notaffect the PGM phenotype observed after elec-trophoresis (data not shown), and this phenotypewas stable throughout plant development, beingdiscernible in stem and petal as well as leaf tissue.Under the stated conditions for electrophoresis,the slow form of the cytosolic PG M isozyme ex-hibits a mobility very similar to the fast allozymeof PGM-2 (see discussion in Weeden and Got-tlieb11)- This overlapping of forms may lead todifficulties in the interpretation of zymogramswhen both Pgm-c and Pgm-p are segregating.We have found that continuing the electropho-resis operation until the borate front has migrated12-13 cm (requiring approximately 5 hours) willhelp resolve the individual bands and facilitateinterpretation of the phenotypes.The method of analysis of the F3 data intro-duced a slight bias favoring the homozygousdominant phenotypes. We did not correct for thisbias in our calculations of chi-square values(Table I) or the recombination fractions (TableII). Correcting for the bias would increase thenumber of wild-type (+) F3 plants in Table I(thereby decreasing the chi-square value). Sim-ilar corrections in Table II would have slight ef-fects on the distribution of progeny among thecategories. However, the calculated map dis-tances would exhibit only minor changes, and therelative order of the loci would not be affected.References1. BLIXT, S. The pea. In Handbook of Genetics, vol.2, R. C. King, Ed. Plenum Press, NY. p. 181 -221.1974.2. INGRAM, V. M. Gene mutations in human hae-moglobin: the chemical difference between normaland sickle cell haemoglobin. Nature 180:326-328.1957.3. LAMPRECHT, H. The variation of linkage and thecourse of crossingover. Agri Hort. Genet. 6:10-48.1948.4. MARX, G. A. and R. PROVVIDENTI. Linkagerelations of mo. Pisum News/. 11:28-29. 1979.5. RICK, C. M. and J. F. FOBES. Association of anallozyme with nematode resistance. Rep. TomatoGenet. Coop. 24:25. 1974.6. SCHROEDER, W. T. and R. PROVVIDENTI.Evaluating Pisum satioum for resistance to peamosaic. New York State Agr. Exp. Sta. Bui. 806.1964.7. and . A common gene for resistanceto bean yellow mosaic virus and watermelon mosaicvirus 2 in Pisum sativum. Phytopathology 61:846-848. 1971.8. SHAW, C. R., F. N. SYNER, and R. E. TASHIAN.New genetically determined molecular form oferythrocyte esterase in man. Science 138:31-32.1962.9. STUBER, C. W., M. M. GOODMAN, and R. H.MOLL. Improvement of yield and ear number re-sulting from selection of allozyme loci in a maizepopulation. CropSci. 22:737-740. 1982.10. TANKSLEY, S. D., H. MEDINA-FILHO, and C. M.RICK. The effect of isozyme selection on metriccharacters in an interspecific backcross of to-mato—basis of an early screening procedure. TAG60:291-296. 1982.11. WEEDEN, N. F. and L. D. GOTTLIEB. The ge-netics of chloroplast enzymes. J. Hcred. 71:392-396. 1980.12. and G. A. MARX. Chromosomal locationsof twelve isozyme loci in Pisum sativum. J. Hered.75:365-370. 1984.13. YEN, D. E. and P. R. FRY. The inheritance ofimmunity to pea mosaic virus. Australian J. Agr.Res. 7:272-281. 1956.412 The Journal of Heredity Notes "^

An isozyme marker for resistance to bean yellow mosaic virus in Pisum sativum

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