Genetic Studies of Induced Mutants in Melilotus alba. II. Inheritance and Complementation of Chlorophyll-deficient Mutants

Six ethyl methanesulfonate-induced mutants of Melilotus alba Desr. were studied. Five of the mutants behaved as monogenic recessives. In the sixth mutant, two independent recessive alleles were responsible for the observed phenotype. Of these two genes, one had a phenotypic effect similar to the five single-gene mutants. The other (the veined gene) caused a chlorophyll deficiency in which the leaf veins were darker in color than the tissue between the veins. Complementation analysis revealed that five of the seven genes detected iu the mutants were nonallelic. Suggested designations for the five genes are ch" ch6, ch., ch7, and chv. Additional index words: Sweetc1over, Ethyl methanesulfonate. G ENE mutations influencing the green coloration of photosynthetically active parts are among the most common spontaneous or induced alterations arising in higher plants. These are usually referred to collectively as chlorophyll mutants. They range from lethal to semilethal or completely viable types having white, yellow, or pale green leaves and stems. Many of the lethal mutants would be of great value in biosynthetic studies, but only rarely is a lethal mutant found whose defect can be corrected by altering the cultural conditions. Consequently, most investigations of chlorophyll mutants in higher plants have utilized nonlethal types capable of growth and reproduction when planted in soil in a growth chamber, greenhouse, or in the field. Knowledge of the 1 Contribution from the Crops Research Division, Agricultural Research Service, U. S. Department of Agriculture, and the Ne· braska Agricultural Experiment Station, Lincoln, Nebraska. Sup· ported in part by the National Science Foundation (Grants Nos. GB·1l48 and GB-8280). Published with the approval of the Di· rector as Paper No. 2639, Journal Series, Nebraska Agr. Exp. Sta. Most of the data were taken from a thesis submitted by the senior author to the University of Nebraska in partial fulfill· ment of the requirements for the M.S. degree. Received Sept. 11, 1969. 2 Formerly graduate assistant in Agronomy, University of Nebraska (now graduate student in the Agronomy Department, University of Illinois); Research Geneticist, Crops Research Di· vision, ARS, USDA; and Bert Rodgers Professor of Agronomy, University of Nebraska, Lincoln, Nebr., 68503, respectively. GEGENBACH ET AL.: INDlJCED MUTANTS IN MEULOTUS ALBA 155 inheritance of such mutants enhances their usefulness in biochemical studies. Interspecific hyhridization within the genus Melilotus results in many chlorophyll-deficient hybrids (6), but most of these have not been studied genetically. The only hybrid subjected to detailed genetic analysis arose from the cross of .lvI. alba Desr. and 1.'11. dentala (Waldst. & Kit.) Pel's. (7). Chlorophyll-deficient Fl hy~r~ds were preserved by grafting to normal M. ottzcznalis (L.) Lam. plants, and were used as the female parents in back crosses with }vL alba. Effects of several nonallelic genes controlling chlorophyll deficiency were noted in progenies of the resulting backcross plants. Three of these genes, identified as chI> Ch2' and Ch3, produced chlorophyll-deficient plants when homozygous, as well as in certain heterozygous combinations. The triply heterozygous condition was lethal in the seedling stage. Subsequent work suggested that chlorophyll deficiency in the Fl hybrid of the M. alba X !vI. dentala cross resulted from the interaction hetween nonallelic genes of the two species (7). Clarke (2) described two spontaneous chlorophylldeficient mutants of 1'11. alba that were semilethal in the greenhouse, but almost 100% of the mutant plants died under field conditions. Each mutant was inherited as an independent monogenic recessive resulting in the production of pale green seedlings. The two genes were designated Pgl and Pg2. A.rece~t report from this laboratory (4) deals with the lsolatIOn of several types of mutants in an annual strain of M. alba, following seed treatments with ethyl methanesulfonate. Numerous chlorophyll-deficient mutants were detected. Many of the deficiencies were lethal, but some mutant lines could be successfully propagated in growth chambers or the greenhouse. Six of these viable, chlorophyll-deficient mutants were available for study when the present work was initiated. The studies reported were conducted to determine the mode of inheritance of these six mutants and to inv~stigate the possihility of allelism among the genes lIlvolved. MATERIALS AND METHODS The chlorophyll-deficient lines were derived from the same source and grown under essentially the same conditions as the morphological mutants previously described (3). All parental, Fj , and F" plants were grown in growth chambers while the F2 progenies were grown in the greenhouse. Characteristics of the parental lines are given in Table 1. The system used for color classification was as follows: #1 albino, lethal; #2 yellow, may be lethal; #3 yellow-green; #4 light green; #5 dark (normal) green. The letter "v" associa ted with a color class denotes the presence of a prominent leaf venation which was considerably darker in color, particularly in the early stages of growth, than the leaves of the #3 or #4 mntants in which it appeared. The veined mutant was readily distinguished from all other mutants at all stages of growth in the growth chambers as well as the greenhouse. The five non veined mutants could Table 1. Characteristics of six chlorophyll·deficient mutant lines and the normal green control. Color Height at Seed Line class Color maturity. em pr~ Q839 ~ Yellow-green ;:]0-.18 Good Q84,1 4 Light green 1.'5-25 l)oor QS44 4 Light green 20-30 eoad Q851 4 Light green :W-::lS Good Q856 4 Ught greer: :~0-:~8 Fair Q858 :1V Yellow-green and vC'inC'c1 80-88 Good Q525 ., ~onn!ll green ~l5-50 Fx('t'llt'nt --------~---.-------."-----------"-be distinguished from each other only during the first 2 to 3 weeks of growth in the growth chamber. All mutants were easily distinguished from the normal control at all stages of growth. Th.e crossing procedure was identical to that previously described (3) except that an attempt was made to obtain seed from all normal X mutant crosses and their reciprocals. For complementation studies, crosses among the six mutants were made in all possible comhinations. but no attempt was made to obtain all possible reciprocal crosses. The 1', plants obtained from each cross were compared to plants of the parental lines with respect to leaf color and venation, height, general vigor, and seed set. For each mutant, 100 to 200 1'2 seeds, obtained by self-pollination of one to three F, plants, were planted to obtain F" segregation ratios. Data from F" progenies segregating for the same phenotype were pooled. The progenies of several F2 plants classified as recessive were planted to determine whether the recessive condition bred true, and the progenies of at least 19 1'. plants of the dominant phenotype also were checked for segregation. In this progeny testing, either 18 or 27 seeds of each dominant F. plant were used, depending on whether the F3 families would be expected to scgrf~gate into two or four classes, respectively. RESULTS AND DISCUSSION Crosses Between Normal and Mutant Lines F t plants were obtained from 10 of the 12 possible reciprocal combinations of the six mutant lines crossed with normal plants. All Fl plants were phenotypically similar to the normal parent in leaf coloration, plant height, and vigor indicating that all six mutants were conditioned by recessive genes. Ratios of normal to chlorophyll-deficient plants in the F 2 and segregating F3 progenies from five of the six mutant lines were approximately 3: I (Table 2), suggesting a monogenic inheritance with normal leaf color completely dominant over each chlorophyll-deficient mutant. Phenotypically mutant F2 plants from each of these five lines bred true in the F3 generation. F2 and some Fa progenies from the sixth mutant line, Q8.58, segregated into four distinct classes as follows: dark green with normal veins (#5), light green with normal veins (#4), light green with dark green veins (#4v), and yellowgreen with light green veins (#3v). The F2 ratio observed gave a satisfactory fit to a 9:3:3:1 ratio, indicating that two recessive genes were apparently responsible for the chlorophyll deficiency and venation observed in line Q858. The conclusion is reasonable that plants of color class #4 resulted when one of the gene pairs was in the homozygous recessive condition; #4v plants were produced when the other gene pair Table 2_ Chi-square analyses of F2 and F3 segregating populations from crosses of six chlorophyll-deficient mutants to normal plants. :\tutant Gener_ ~o. of Distribution among color classes line ation* families #,5 #4v #4 #3v #3 Q839 F~ (5) 3 1,53 46 F, (,5) 14 149 41 Q843 F, (,5) 132 44 r30) 1,1 118 40 QR4·1 Fz (.') 141 51 F~ (.'») 1.5 149 :il Qa,51 Fz (5) 81 20 }~ (.'5) 1.1 167 D1 QSS6 r~ (5) 76 22 F~ (5) 11 10:3 2G Q8.'58 F, (5) 57 20 22 Fj (.5) S.'5 2.s F3 (.'5) 2 2·j 8 F',,(.,) ]2 12:1 40 29 10 1-'" (4v) 1.5 1:l8 49 F; (4) .')1 1,8 of-flt to :l 9<"1::1, L ratio; all others lp~tpd Chip