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MUTANTS AND LINKAGE I N MORMONIELLA GEORGE B. SAUL ~ N DAND MARION KAYHART Dartmoath College, Hanover, New Hampshire and Cedar Crest College, Allentown, Pennsylvania Received July 27, 1956 HE research of various workers has resulted in the accumulation of a large number of mutant stocks of Mormoniella vitripennis (WALKER).Some of these mutants have been described by WHITING(1951); more recently, many others have arisen spontaneously or as the result of radiation studies conducted by KAYHART (1956) and SAUL(1955). Many of the mutants are a t the complex locus which WHITING(1951) has named the R locus, but mutations a t other loci have also been found. Because of the increasing use of Mormoniella as research material, it is desirable to describe these mutants, and to summarize their linkage relationships. It would further be desirable to have available a linkage map corresponding to the five chromosomes of Mormoniella; this study is a contribution toward that goal. T MATERIALS AND METHODS Mormoniella is a chalcidoid Hymenopteron parasitic on the pupae of various muscoid Diptera. Males are haploid and arise from unfertilized eggs; diploid females arise from fertilized eggs. Unmated females thus produce only male progeny; the ratio of phenotypes among these approximates the segregation of genes among the gametes of the parent females. The blowfly Sarcophaga bullata Parker is used as the host in the experiments to be reported here. Details of the life cycle of Mormoniella are described by SAUL(1955). The wild type wasps have reddish-brown eyes and iridescent bronze-colored bodies. Females have darker antennae and body color than have the males. The wings of the females are of normal size; the males are brachypterous. Most of the mutations obtained to this time affect either eye color or body color. The mutant eye colors vary from black through deep red, orange-red, orange and light peach, to oyster-white. In general, double mutants are in color as light as or lighter than either single mutant type. Body colors include purple, various bluegreen shades, and deep blue. A lavender body color mutant was obtained, but it was lost before it could be tested for linkage. Mutants are kept as pure stocks unless they are female-sterile, in which case heterozygous females are crossed with mutant males. All of the mutants are recessive to wild type. In this study, standard conventions are followed in designating the mutants and previously published names of mutants are retained. Eye color mutants for which a name has not been published are named, when possible, by reference to colored plates in A Dictionary of Color (MAERZand PAUL1950). The number of the plate which corresponds most closely to the eye color is used in the description of the mutant, and where possible the name attached to the plate is used for the mutant. Other mutants, such as body color mutants, which can not be named by this method, are named arbitrarily. MUTANTS A M ) LINKAGE I N MORMONIELLA 93 1 The apparatus used for comparing eye colors with the Maerz-Paul color charts was designed by DR. SARAH FLEMISTER. The wasp is observed by reflected light under the low-power objective and 1OX ocular of a compound microscope. It is placed on its side, with the surface of the eye in full view. The color charts are covered with a piece of black cardboard which has an opening in the center; this opening permits comparison of the eye color with one tab at a time. A spotlight illuminates the eye and a second spotlight illuminates the color chart, which can be observed by shifting one's view only slightly from the microscope. All observations were made on freshly eclosed males. Linkage tests were made by two factor crosses. I n general, five males of one mutant type were crossed with five females homozygous for a second mutant gene. Five F1 females from the cross were set unmated and the Fz progeny, all male, were counted. Linkage, when present, was shown by reductions in the numbers of wasps in the recombinant classes (wild type and, in the absence of epistasis, double mutants). If both mutants to be crossed were female sterile, PI females heterozygous for one of the mutant types were used. Each F1 female was set individually and only those Fz cultures which contained both mutant types were used for linkage counts. These cultures would be expected to include about half of the total number set. If a cross indicated that two traits were linked, then, when possible, five males of the double-mutant type were crossed with five homozygous wild type females and the cross was carried to the FZgeneration as described above. In these cases linkage was thus estimated from crosses in the coupling and in the repulsion phases. When possible, the calculation of linkage followed the geometric mean averaging system given by FISHER in The Design of Experiments (1949). The gametic ratio r / s is given by: In this formula, a1 and a2 are the total recombinants from the coupling and repulsion experiments respectively; bl and bz are the total parentals from these crosses. This method compensates for differences in viability of the classes, but does not compensate for linked lethals. In the case of epistasis, linkage was estimated from the cross in repulsion phase by dividing the number of wild type wasps (m) by the total number of wasps in the wild type class and the class ( n ) showing the hypostatic trait. The formula is thus m / ( m n ) . The formula n/(m n) gives an estimate of linkage from the cross in the coupling phase. Since the double mutants could not be selected with certainty, several crosses were made for the coupling experiment. Each mating involved a single male from the class showing the epistatic trait and a wild type female; only Fz cultures which included wasps showing the hypostatic trait (and which thus were sired by double-mutant males) were used for linkage counts. The geometric mean averaging system employed for more precise measurement of linkage can, in this case, use only one class of parentals and one of recombinants from each cross, since the other classes are identical due to the epistasis. In the general formula given above, + + 932 GEORGE B. SAUL ~ N D AND MARION KAYHART al, a2 ,bl , and bz represent only the distinguishable parental and recombinant classes; the method thus becomes the same as that proposed by MULLER(1916). In Fz cultures from some crosses, only wild type and one class including both the single-mutant and the double-mutant types could be distinguished. In these cases, linkage was measured by doubling the number in the wild type class and dividing it by the total count in the culture. MUTANTS The following list is a brief summary of information about 22 mutant types of Mormoniella. Apparent isoalleles are not included, nor are mutations that were lost before they could be tested and characterized adequately. Important information about the factors scarlet-DR, tomato, garnet, and purple has been reported by WHITING(1951) ; these factors are therefore listed here with only the Maerz-Paul references (where applicable) and literature citations. Of the numerous alleles a t the complex eye color locus R, only those used in linkage tests are listed. The following information is given for all mutants except those mentioned above: symbol, name, investigator who found the mutant, date found, inducing agent (if any), description and important characteristics, and Maerz-Paul reference (if applicable). The MaerzPaul reference includes the plate and tab numbers. bk: black. GROESBECK1952. Induced by X-rays. Eyes black, body slightly greener than wild type. Interacts with dark red eye colors to give lavender or light peach and with scarlets and lighter colors to give white. 48:L-12. bl: blue. KAYHART 1953. Spontaneous in wild type stock. Body dark blue. Female sterile. Viability considerably lower than wild type. ga: garnet. 3:L-11. (WHITING1951). g@8t-b:garnet-scarlet-b. KAYHART 1953. Induced by fast neutrons. Eyes orange-red, phenotype identical to R s t - D R 2 :L-11. gZ: glass. MILROOD 1952. Spontaneous. Eyes narrow, facets poorly differentiated and reduced in number. Female almost sterile. Viability somewhat lower than wild type. gr: green. KAYHART 1953. Induced by slow neutrons. Body grass-green. Female sterile, viability somewhat lower than wild type. gb : green-blue. WHITING1952. Spontaneous from wild type. Body greenish-blue to deep blue. Female sterile. or: orange. SAUL1952. Induced by X-rays. Eye color dull orange. 2:D-12. pZ: pellucid. WHITING1952. Spontaneous from +/ti-277. Eye color oyster-white, phenotype identical to R o U - D R . Epistatic to all other eye colors. 4:A-7. pu: purple. (WHITING 1951). rh: reddish. KAYHART 1953. Induced by slow neutrons. Eye color dark rust-red. 6: 1-11. R 8 t - 4 - D R : Scarlet-DR.2 :L-11. (WHITING 1951). scarlet 426. GROESBECK1952. Induced by X-rays. Phenotypically identical to scarlet-DR.2 :L-11. st-c: scarlet-c. WHITING1953. Spontaneous from wild type. Phenotypically identical to scarlet-DR.2:L-11. 933 MUTANTS AND LINKAGE I N MORMONIELLA TABLE 1 Itdependent assortment of traits in Mormoniella, including data from representative two-factor crosses. Genes are listed alphabetically; crosses of each gene, in order, with succeeding genes are grouped together. A dash in the list of FZ classes from a cross indicates that the class i s included with another class identical or epistatic to it. The symbol a/b means that the FI female is heterozygous for factors crossed in repulsion phase; ab/+ indicates factors crossed in coupling Fz males FIfemale from cross a X b or ab X + + 72 41 98 40 76 49 71 69 54 68 114 72 71 95 65 80 38 60 56 118 103 84 99 40 109 96 96 69 100 157 44 51 35 78 77 54 60 133 75 62 100 45 a 87 39 99 61 79 53 75 61 60 118 103 63 84 93 56 50 32 66 56 90 78 80 103 38 95 94 95 80 87 138 44 53 31 66 73 78 62 111 66 59 92 47 b 58 29 83 39 79 119 77 58 62 72 107 74 73 65 59 37 81 45 104 85 78 76 97 212 110 97 74 ab (with phenotype) 56 13 pale red 66 57 95 oyster 62 deep gentian 56 oyster 57 oyster 81 oyster 124 pale red 55 dark red 85 61 65 30 70 46 63 99 79 oyster 90 scarlet 87 oyster 73 oyster 72 scarlet 39 55 31 97 73 54 59 32 55 24 60 51 61 4: 58 57 75 52 78 57 97 44 934 GEORGE B. SAUL 2ND AND MARION KAYHART TABLE l.-Contimed FI female from cross a X b or ab X + A males + 75 60 472 63 105 42 109 53 59 117 78 92 86 56 65 178 86 53 94 217 154 76 93 so 87 135 109 69 76 167 56 109 106 67 85 138 a 68 61 408 67 107 38 90 120 119 99 120 137 187 155 173 166 83 54 124 196 157 224 283 81 96 124 307 76 73 149 70 79 114 70 98 133 b 78 73 154 88 45 84 59 85 89 85 70 64 86 56 109 353 86 72 ab (with phenotype) 54 69 60 oyster 54 oyster 70 light peach 29 white - - 81 orange 54 orange 122 orange - - 96 light peach 70 orange 56 72 79 oyster 77 orange 73 71 61 light peach 73 orange 67 43 oyster st-d: scarlet-d. KAYHART 1953. Induced by slow neutrons. Phenotypically identical to scarlet-DR.2:L-11. tl: tile. KAYHART 1953. Induced by X-rays. Eye rust-red. 3:D-12. t b g - b : tile-oyster-b.,WHITING1954. Spontaneous from wild type. Eye color oysterwhite; phenotypically identical to pl and epistatic to all other eye colors. Complementary to tl. 4: A-7. to: tomato. 4:F-12. (WHITING1951). tota tomato-tanagra. SAUL1952. Induced by X-rays. Dark red eye color approaches wild type with increasing age. Female sterile. Viability low. 7 :J-9. 935 MUTANTS AND LINKAGE I N MORMONIELLA tomato-manzanita. WHITING1953. Spontaneous from wild type. Eye color dark red, slightly browner than tota in color. 7:L-10. tomo: tomato-moroccan. KAYHART 1953. Induced by slow neutrons. Eye color dark red, but lighter than tota. 5:K-11. tomato-moroccan-2. KAYHART 1953. Induced by slow neutrons. Eye color overlaps tomDin adults but is slightly lighter in pupae. Wild type: 8:H-8. tomz: RESULTS O F LINKAGE TESTS As shown in the preceding list, 15 loci are now recognized in Mormoniella. Mutants a t 14 of these have been tested for linkage; gr has not been tested due to difficulties in establishing it in stock. For similar reasons bl has not been crossed with genes a t all known loci. Table 1 gives results of tests which show no linkage; Table 2 gives results of tests which indicate that the genes crossed are linked. In the left-hand columns of the tables are genotypes of FI females from the crosses of each gene with each succeeding gene: a/b means that the female is heterozygous for a and b and that the cross was in repulsion, whereas ab/+ means that the cross was made in coupling. The four columns of Fzmales are arranged to show the wild type (+), mutant type a, mutant type b, and the double mutant ab. Phenotypes of double eye color mutants are given when distinct from either single mutant type. I n most cases, only one mutant a t a locus was used to test the locus for linkage. This mutant was selected on the basis of high viability and a phenotype easily distinguished from wild type and (if possible) from other mutants used in the crosses. A few additional crosses, not included in the tables, indicated that linkage values were unchanged when other alleles a t linked loci were crossed; the one exception to this (alleles a t the Ga locus) is reported and discussed below. TABLE 2 Linkage of traits in Mormoniella. See legend of table I for explanation of organization of table FI female from cross a X b orab X + bl/gb ga/tl ga tl/+ ga*t--b/tl gaa$-btl/+ gl/Ra'-DR gb/rd gb/tomz gb tomz/+ or/st-c or st-c/+ pllst-d pl st-d/+ rd/tomr rd tomr/+ F2 males ! + 51 106 124 21 160 4 28 84 190 21 200 65 97 15 220 a 321 20 155 17 247 165 240 50 290 7 317 110 - b 244 357 22 172 22 230 158 200 42 2 78 4 261 20 547 9 ab (with phenotype) 88 oyster 95 oyster 17 light peach 182 light peach 8 41 84 149 14 light peach 159 light peach - 5 light peach 122 light peach 936 GEORGE B. SAUL 2ND AND MARION KAYHART The data in the tables indicate that there are five groups of linked factors and a single locus (Bk) not included in any of the linkage groups. When linkage is calculated as described above, the groups are as follows: 1. R Pu 10.6 1-24.-1 2. To GI Rd 3.1 1-24.4-----1 Gb 17.6 BI 34.6 or 3. st-c 4.2 St-d 4. PI 18.5 5 . TI Ga (19.0 or 10.3) 6 . Bk Distances are in Morgan Units. Rd and B1, and To and B1 recombined a t random. The R-Pu linkage was calculated from the data of WHITING(1950). If ga is crossed with tl, 19.0% recombination is observed; if is used in the cross, 10.3% recombination results. These percentages of recombination are almost unchanged if other alleles a t the TI locus are used in the crosses. It is therefore possible that the gaat-bstock contains a crossover suppressor linked with the Ga locus. The cross of tlou-b with tl gives wild type F1 females; these females, unmated, produce only tlo’-b and tl Fz males. It is possible that tlou-b and tl are a t separate, completely linked loci. I t is also possible that they are at one complex locus and show the complementary allelism that has been described for the R locus. This second possibility is the basis of the nomenclature adopted here. The factor for purple body color shows about 10.6% recombination with the R locus if the wild type allele for glass eye is present. When gl is present, the R-Pu recombinations drop to about 2.1 % (19 in a culture of 897). The R locus and glass show about 2.4% recombination; recombinations between gl and pu occur in less than 1% of the possible cases. It is suggested by DR. P. W. WHITING(1955) that “gl” may be an inversion, and that the Pu locus may be included within the inversion. Cytological observations on gl/+ females have not yet been made. The viability of gl males is about equal to that of males, and SI/+ females have high fecundity. The fecundity of homozygous gl females is very low, however. Spermatogonial cells of Mormoniella contain five chromosomes, the haploid number for the species. Although this study has shown the existence of five linkage groups and one factor, black, segregating independently of all other loci, it is impossible to determine a t present whether the five linkage groups are located on five separate chromosomes. + MUTANTS AND LINKAGE IN MORMONIELLA 937 SUMMARY 1. New mutants in Mormoniella are described and discussed. A system for describing and naming eye color mutants is introduced; this system is based on the use of Maerz-Paul color charts. 2. The known loci are shown to be associated in five linkage groups as shown on page 936. 3. Data obtained by the use of gl stock are discussed. These data support a suggestion by WHITINGthat “glass” is an inversion which may include the Purple locus. 4. It is possible that the Tile locus is complex, containing factors which show complementary allelism. ACKNOWLEDGMENTS Much of this study was completed while the authors were graduate students a t the University of Pennsylvania. During this period the work was supported by the National Institutes of Health (G. B. S., predoctoral fellowship) and the Atomic Energy Commission (M. K., assisting DR. P. W. WHITINGunder contract AT(30-1)-1471 of the University of Pennsylvania with the U. S. Atomic Energy Commission). LITERATURE CITED FISHER, R. A., 1949 The Design of Experiments. New York: Hafner Publishing Company, Inc. 5th ed., pp. 221-225. KAYHART,M., 1956 A comparative study of dose-action curves for visible eye-color mutations induced by X-rays, thermal neutrons, and fast neutrons in Mormoniella vitripmnis. Radiation Res. 4: 65-76. 1950 A Dictionary ojColor. New York: McGraw Hill Book Company, MAERZ,A., and M. R . PAUL, Inc., 2nd ed. MULLER,H. J., 1916 The mechanism of crossing over. Am. Naturalist 60: 352-354. SAUL,G. B. ~ N D ,1955 The induction by X-rays of recessive lethals in the mature sperm of Mormoniella vitripennis (Walker). Radiation Res. 2: 447460. WHITING,P. W., 1950 Linkage in Mormoniella. Genetics 36: 699. (Abstract) 1951 Multiple complementary alleles in Habrobracon and Mormoniella. J. Genetics 60: 206214. 1954 Comparable mutant eye colors in Mormoniella and Pachycrepoideus (Hymenoptera: Pteromalidae). Evolution 8: 135-147. 1955 Linkage relations of purple, glass, and the eye-color locus R in Mormoniella. Genetics 40: 602. (Abstract) WHITING,P. W., and L. H. BENKERT,1934 Azygotic ratios in Habrobracon. Genetics 19: 237-267.