Báo cáo khoa học: "Genetic markers for Prunus avium L: inheritance and linkage of isozyme loci"

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Original article Genetic markers for Prunus avium L: inheritance and linkage of isozyme loci F Santi* M Lemoine B Bruant INRA, Station d’Amélioration des arbres forestiers, Centre de recherche d’Orléans, Ardon, 45160 Olivet, France January 1990) 1 February 1989; accepted 14 (Received Summary - The polymorphism of 10 enzyme systems in wild cherry (Prunus avium L.) was analysed using vertical polyacrylamide gel electrophoresis (AMY, GOT, ME) and isoelectro- focusing (ACP, IDH, LAP, MDH, PGM, SDH, TO) on 286 wild cherries. The products of around 41 loci could be distinguished in these systems, 13 of which displayed polymorphism. The genetics of 7 isozyme loci have been studied using 8 fullsib families: acp1, lap1, sdh1 and as monomers, and got1, idh1 and me1 were active as dimers. Two other mdh1 functioned isozymes (pgm1 and got2 ) appeared to have simple inheritance, but it was not possible to verify this. Joint segregation of 13 locus pairs showed linkage between lap1 and got1 (r = 0.05 ± 0.07). ldh1, sdh1, acp1 and mdh1 are 0.03 ± 0.02) and between lap1 and me1 (r = not linked to these loci. No linkage has been detected between acp1 and mdh1. linkage / inheritance / variability / Prunus avium L. / wild isozyme / cherry Résumé - Marqueurs génétiques pour Prunus avium L : déterminisme génétique et groupes de liaisons entre loci enzymatiques. L’espèce Prunus avium comprend les cerisiers, variétés améliorées pour la production de fruits, et les merisiers, arbres forestiers de qualité sur lesquels portent aussi des programmes d’amélioration. Il serait utile pour ces programmes de disposer de marqueurs génétiques, pour les identifications clonales et interspecifiques, l’analyse de la variabilité naturelle et du système de reproduction et le contrôle des produits de croisements dirigés. Comme peu de marqueurs génétiques avaient été étudiés chez P avium, nos efforts ont porté sur la recherche et la caractérisation de loci enzymatiques. Le polymorphisme enzymatique a été étudié grâce à 286 merisiers provenant de France (216), d’Allemagne (14) et de Belgique (6). Le déterminisme et les liaisons génétiques ont été testés avec 8 descendances d’un demi-diallèle 14 x 14. Les extraits de bourgeons prélevés en hiver ont été analysés par electrophorèse sur gel d’acrylamide (AMY, GOT, ME) ou par isoélectrofocalisation (ACP, IDH, LAP, MDH, PGM, SDH, TO). Dans les zymogrammes obtenus, schématisés en figure 1, 13 loci enzymatiques polymorphes et 28 bandes monomorphes 2 sont observés. Les hypothèses de déterminisme génétique (fig 1) ont été testées par un χ dans les descendances (tableau II). Deux écarts significatifs (au niveau 5 %) aux proportions * Correspondence and reprints
attendues par ségrégation génotypiques mendélienne ont été notés, mais le test de 2 χ global est non Le déterminisme de acp1, got1, idh1, lap1, mdh1, me1 et significatif. sdh1 a ainsi été établi. Un seul type de test de déterminisme était possible pour amy1, mdh2, got2, pgm1et to1, car les 14 parents du demi-diallèle ont les mêmes génotypes supposés (homozygotes pour les 4 derniers loci). Cela n’a pas permis d’établir avec certitude le déterminisme proposé. Cependant, en comparant le type de zymogramme obtenu par d’autres auteurs sur les mêmes enzymes, il semblerait que le déterminisme le plus probable pour got2 et pgm 1 soit celui proposé. La coségrégation de 13 paires de loci a été testée par rapport à celle attendue en cas d’indépendance (table III), montrant une liaison signifi- cative entre got 1 et lap 1 (r = 0.03 ± 0.02), et entre lap 1 et me 1 (r = 0.05-0.07). Ce groupe de liaison est indépendant de idh 1, sdh 1, acp 1 et mdh 1 et aucune liaison ne semble exister entre ces 2 derniers loci. Toute autre relation entre loci n’a pu être testée avec les familles analysées. Prunus avium L / merisier / / déterminisme isozyme / liaison génétique INTRODUCTION leles, among sweet cherry cultivars (Crane and Brown, 1937), as well as The sweet cherry, Prunus avium L, is among wild cherry (Berger, 1963, Santi, widely cultivated for its fruit crop, for unpublished data), but it is necessary which substantial improvements have to make crosses for the genotype de- been made for some time. The same termination. species, named the wild cherry, grow- Treutter and Feucht showed (1985) ing naturally in Europe and West-Asia, differences in phenolic composition produces a very valuable wood, justi- among P avium clones. Phenolic com- fying the forest breeding programmes pounds are potentially valuable genetic which began recently. markers for breeding programmes and Genetic markers would be useful for population genetic studies, since they both fruit and wood breeding pro- may be linked directly to economically grammes. The identification of varie- important traits (Doumanjou and ties, control of breeding material, and Marigo, 1978; Friend, 1985) and involve the assesment of specific purity would regulator genes (Vernet et al, 1986). be possible with a series of en- Unfortunately, phenolics are often af- vironmental influence-free traits. Co- fected by environmental factors and dominant, simply-inherited and mapped their inheritance is complex. traits are useful for various purposes, Such difficulties do not usually arise such as the analysis of natural varia- while using isozymes, the most widely bility and of mating systems, the control used genetic markers, which have al- of man-made crosses and of products ready proved useful for numerous of forestry clonal seed orchards. plants as reviewed by Tanksley and Very few monogenic, simply-inherit- Orton (1983). Some knowledge is now ed morphological traits have been de- available on P avium enzyme variability. scribed for Prunus avium: fruit-juice Feucht and Schmid (1985) showed pro- colour and albinism, which are control- tein and peroxidase banding pattern led by dominant-recessive pairs of al- differences among P avium clones. leles (Watkin and Brown, 1956). Kaurisch et al (1988) pointed out vari- The gametophytic incompatibility S ability for 3 isozyme loci (aconitase-2, locus is polymorphic with at least 6 al- 6 phosphoglucodeshydrogenase-1,
tion does not alter enzyme activity, since the phosphoglucoisomerase-2). Neverthe- extracts of fresh and lyophilized buds of 5 less, the number of enzymes studied is clones had similar electrophoretic patterns. still limited and no inheritance has been 100 mg of scale-free buds were put into established. In addition no linkage map an aluminium bag, frozen in liquid nitrogen of P avium is available. and crushed with a hammer. The powder The purpose of this study therefore was soaked for 1 h in 1.2 ml of extraction buffer (20 mM triscitric pH = 7.5 containing was to increase the number of isozyme 12 mM 2β-mercaptoethanol, 5 mM dithi- loci available for P avium and to test otreithol, 2 mM polyethylene glycol (MW = their inheritance and linkage relation- 6 000), 2% w/v PVP). When less than ships. 100 mg of buds were available, the buffer volume was adjusted. The homogenate was centrifuged for 30 min at 14 000 g, and the supernatant, transferred into plastic vials, MATERIAL AND METHODS was stored at -60 °C until electrophoresis. Plant material Electrophoretic procedure Enzyme variability was studied with 286 wild cherries sampled throughout most of France polyacrylamide gel elec- The trisboric-EDTA (186) and in 4 populations in: Normandy trophoresis (Page) system (Dalet and Cornu, (Northwest France, 61 trees), the Ardennes 1989) was performed for 3 enzymes. Gluta- (Northern France 19 trees), Southern Germa- (GOT, EC mate oxaloacetate transaminase ny (14 trees) and Southern Belgium (6 trees). 2.6.1.1.) was run through a 10% acrylamide The inheritance and linkage analyses "running gel" and a 5% acrylamide "stacking were made with 1 year-old plants of 8 fullsib gel". Malic enzyme (ME, EC 1.1.1.40) and families, which were chosen in a 14 x 14 amylase (AMY, EC 3.2.1.1) were run through half-diallel according to the availability of 7% and 10% acrylamide "running gels", res- material and the variability of parent isozyme pectively. The power supply was set at 100 phenotypes: nr13 (clone 108 x clone 229), V for 1 h, then bromophenol blue and 15 μl nr22 (109 x 208), nr27 (111 x 143), nr36 (GOT) or 20 μl (ME and AMY) of extract (111 x 229), nr71 (171 x 195), nr80 loaded into each slot. The voltage was were (195 x 226), nr81 (195 x 229), nr87 then increased to 250 V and maintained for (208 x 226). 5 h (AMY) or increased to 350 V and main- Buds were preferred to leaves for enzyme tained for 6 h (GOT, ME). The temperature extraction. Buds were sampled during the of the electrolytic buffer was kept at 4 to 1987-1988 winter. The samplings were made 6 °C using a cooler. in the original stands in Normandy and The other enzymes were run on on 15 to 100 year-old trees. The Bavaria 245 x 125 x 0.5 mm isoelectrofocusing (IEF) samplings of other wild cherries were made gels, containing acrylamide and bisacry- on 1-7 year-old vegetative copies in a clone 5%, C lamide (concentration T 4%), = = bank in Olivet. The sampling period varied Servalyt carrier ampholytes (2%) w/v 3- from November 1987 to March 1988. The 10 pH gradient, 0.7% w/v 4-6 pH gradient; buds were sampled from the lateral or apical the latter was not added when running position, on short or long shoots. The ma- deshydrogenases, 1 μl/ml TEMED and jority of the buds were vegetative, and ex- 1.8 mM ammonium persulphate. The gels ceptionally floral. were run on 2 "Multiphor II" apparatus, with the cooling plates maintained at 2 °C. The electrolytes used were those described by Sample preparation Kinzkofer and Radola (1981). Six to eight μl of extract were loaded using a hand-made silicon applicator strip with 64 holes, lying Immediately after sampling, the buds were across the gel. The power supply was set frozen in liquid nitrogen, freeze-dried and at a maximum of 1200 or 1500 V, 45 mA and vacuum-stored until extraction. Lyophiliza-
40 W for the 2 gels, and each run lasted among the 286 wild cherries, except for ME, about 2 h. for which only the 14 parents of the half-dial- lel and 5 of their progenies were observed. The staining procedures were those of Departure from or adequation to the expec- Cardy et al (1983) for ME, GOT, isocitrate ted segregation ratios in the observed fami- deshydrogenase (IDH, EC 1.1.1.42.), malate tests. &2 chi; lies were tested using deshydrogenase (MDH, EC 1.1.1.37) and phosphoglucomutase (PGM, EC 2.7.5.1.). They were those of Roux and Roux (1981) for acid phosphatase (ACP, EC 3.1.3.2), and RESULTS those of Beckman et al (1964) for leucine aminopeptidase (LAP). For AMY staining, gels were soaked 2:30 h in an acetate buffer Scored loci and inheritance hypo- 0.2 M pH 4.5 with 2% w/v starch and 6% = theses w/v CaCl and then in the same buffer with , 2 3% w/v Kl and 0.3 w/v l PAGE gels were . 2 All the observed zymograms of the 286 fixed with 7% acetic acid, wrapped in plastic wild cherries are represented schemati- foil and stored at 4-6 °C. IEF gels were dried and stored at room temperature. cally in figure 1. Thirteen polymorphic loci and 28 monomorphic isozyme bands were scored. Checking zymogram stability Inheritance hypotheses (figure 1) are easy to propose for acp1, got1, idh1, was tested by varying Zymogram stability the sampling conditions applied to the same lap1, mdh1, me1and sdh1, since at clones as described in table I. No difference least 3 supposed genotypes (aa, bb, was noticed for 9 enzymes (ME stability was ab) appear directly from the observed not tested), ensuring that the observed zy- phenotypes. The pattern of the bb zy- mogram differences were independent of the mogram (nr3 on figure 1) of the got1 tested sampling conditions. locus suggests that a monomorphic band is merging into the a-band. This Inheritance and tests linkage band may be the product of a dupli- cated GOT-locus. For mdh1, one allele Mendelian inheritance hypotheses were pro- is thought to produce 2 bands. Con- posed after watching zymogram variability
versely, only 2 phenotypes of amyl, family n° 22 for the locus acp1. These got2, mdh2, pgm1 and to1 were re- departures are not significant for the corded in observed zymograms. Two total of 25 chi-square tests. Neverthe- hypotheses are proposed for mdh2: the less, a linkage between the acp1 locus product of the more frequent allele is and the incompatibility S-locus may thought to be merged into a monomor- provide such a result for the family 22, phic band; or a null allele is involved. if the parents have 1 common S-allele, The expected heterozygote was in because the male parent is a heterozy- general the least often observed gote for the acp1 locus. phenotype: 1/285 for got2, 35/285 for mdh2, 9/285 for pgm1,24/285 for to1, Joint at locus segregation pairs that the second homozy- suggesting not recorded by chance. gote was Over the 13 locus pairs tested for joint Further difficulties arise when interpret- segregation 2 pairs had highly signifi- ing amyl phenotypes, since the pheno- cant (P < 0.01) chi-square values for type with the most numerous bands is 2-locus segregation ratios. The loci in- the most frequent (241/285). A realistic volved are the pairs got1/lap1 and hypothesis is to involve a null allele. me1/lap1. The recombination fraction Sdh1’ and lap1’ appear as secondary between the loci is reduced in all fami- products and me1’ is probably a het- lies for these 2 pairs, although in the erodimer between me1 and a mono- cross n° 27 the chi-square value is not morphic locus. No simple genetic significant for the me1/lap1 pair. hypothesis results from the examination The estimated recombination values of the acp2 patterns. show a strong linkage between got1 and lap1,and between me1and lap 1, but the precise respective position of at individual loci Segregation the 3 loci is still unknown, since the linkage between got1 and me1 has not The scored monomorphic bands yet been tested. ldh1, sdh1, acp1and among the 286 wild cherries were still mdh1 are not linked to this 3 locus monomorphic among the progenies. group, according to 2, 4, 5 and 2 tests All 14 parents of the half diallel were respectively. The Acp1/mdh1 linkage monomorphic for amyl (thought to be relationship has only been tested heterozygote with a null allele or ho- among the acp1, mdh1, idh1and sdh1 mozygote with active alleles), and for loci, and it involved only 14 sibs. mdh2, got2, pgm1and to1 (thought to be The observed segre- homozygotes). gation ratios in 1 progeny of the 3 latter DISCUSSION isozyme loci were those expected, and for amyl no evidence for a null allele appeared (table II). Inheritance Other loci were polymorphic among A limited number of sibs per family the 14 parents, allowing more diverse were analysed because buds were crossing combinations. The results in most often restricted in number and table II show 2 departures from Men- size. Nevertheless, we have deter- delian expectations, concerning the mined that 7 isozymes detectable in P family n° 80 for the locus sdh1 and the
organisms. GOT avium buds in other (acp1, got1, idh1,lap1, existing usually dimers, and PGM inherited as mdh1,me1,sdh1 ) isozymes are are are usually monomers with single genes, since observed segrega- isozymes tion ratios in several full-sib families are duplicated bands (Pasteur et al, 1987), so the observed pgm 1 and got2 pheno- as expected. Acp1, lap1, sdh1 and types in P avium do not differ from the mdh1 seem to be monomers, while usual patterns, and therefore the pro- got1, idh1 and me1 have dimer-like pat- posed inheritance hypotheses are the terns. These patterns are common in most likely. TO isozymes are not usually animals or plants (Tanksley and Orton, monomers (Pasteur et al, 1987), so 1983, Pasteur et al, 1987), apart from care should be taken with the proposed MDH isozymes, which are usually inheritance. dimers. of alleles No more information will be forth- identifying Conversely, amy1 and mdh2 would be difficult, if coming by analysing more families of the proposed inheritance is correct, the half diallel for pgm1, to1, got2 and since the supposed genotypes do not mdh2, since the 14 parents have the always result in different phenotypes. homozygous genotype. New same Acp2 inheritance is not clear, though crossings, involving variable geno- several progenies have been analysed. types, will be necessary to obtain clear Out of the 13 isozyme loci, 9 may there- evidence of the allelic relationships fore be useful in population genetic among the observed bands of the pu- studies. These are in addition to the 3 tative loci. Nevertheless, one may com- observed by Kaurisch et al (1988), who pare the obtained phenotypes to those
Doumenjou N, Marigo G (1978) Relations proposed inheritance patterns, which polyphenols-croissance : rôle de l’acide although not tested, are the most chlorogénique dans le catabolisme auxi- probable. nique. Physiol Veg 16(2), 319-321 Feucht W, Schmid PPS (1985) Determination of proteins and peroxidases by ultrathin- Linkage relationships layer isoelectric focusing in callus from 4 Prunus species Angew Bot 59, 71-79 has 8 chromosome pairs. P avium Friend J (1985) Phenolic substances and 12 variable isozyme loci Among the plant disease. 367-397. In: Ann proc of with known inheritance, 3 are on the the Phytochemical Society of Europe, vol 25, (Vansumere CF, Lea PJ, eds) same chromosome pair, 4 are not part Clarendon Press, Oxford of this linkage group, but are not nec- Kaurisch P, Gruppe W, Kohler W (1988) essarily on 4 different chromosomes, Enzympolymporphismen bei Kirschen and the remaining 5 loci have unknown (Prunus spp) und Arthybriden (p.x spp). linkage relationships. New analyses will Methode, ausgewählte Arten/Sorten und therefore be necessary to complete the Unterlagen-Reis-Wechselwirkungen. Angew Bot 62, 41-52 linkage map. Miniature ul- (1981) Kinskofer A, Radola BJ The sizeable number of polymorphic trathin-layer isoelectric focusing in 20-50 isozyme loci in wild cherry will prove μm polyacrylamide gels. Electrophoresis useful in our research programme, in 2, 174-183 the breeding programme as well as for Pasteur N, Pasteur G, Bonhomme F, Catalan population genetic studies. J, Britton-Davidian J (1987) Manuel tech- nique et génétique par électrophorèse des proteines. Techniques et documen- REFERENCES tation, Lavoisier, Paris, 217 Roux L, Roux Y (1983) Identification bio- chimique de clones et de lignées Beckman L, Scandalios JG, Brewbecker JL d’asperge (Asparagus officinalis L, Lili- (1964) Genetics of leucine aminopep- acées). Agronomie, 3, 57-66 tidase isozymes in maize. Genetics 50, Tanksley SD, Orton TJ (1983) Isozymes in 899-904 plant genetics and breeding. Elsevier, Berger K (1963) Fertilitätsuntersuchungen an Amsterdam Part A, 516 part B, 472 der Wildkirsche Prunus avium L var avium Treutter D, Feucht W (1985) Art-und klon- Kulturplanze 11, 358-375 des Polyphenolmuster spezifishe Cardy BJ, Stuber CW, Wendel JF, Goodman Phloems von Prunus avium und P cera- MM (1983) Techniques for starch gel sus. Mitt Klosterneuburg 35, 256-260 electrophoresis of enzymes from maize Vernet P, Gouyon PH, Valdeyron G (1986) (Zea mays L.). 35 p, North Carolina State Genetic control of the oil content in thy- University, Raleigh mus vulgaris L.: a case of polymorphism Crane MB, Brown AG (1937) Incompatibility in a biosynthetic chain..Genetica 69, 227- and sterility in the sweet cherry Prunus 231 avium L. J Pomol Hortic Sci 15, 86-116 Watkin W, Brown AG (1956) Genetic re- Dalet F, Cornu D (1989) Lignification level sponse to selection in cultivated plants: and peroxidase activity during in vitro gene frequencies in Prunus avium. rooting of Prunus avium L. CanJ Bot (in Heredity 10, 237-245 press)