Marcus Rhoades, Preferential Segregation and Meiotic Drive

LONG before microarray biologists coined and promoted the term “discovery science,” maize geneticists were avid practitioners of this mode of investigation. In fact, one might say that for a number of years, the field of maize genetics basically operated as discovery science. Many have speculated about why maize remains a model organism for genetic analysis, given its long life cycle relative to other species. It has many virtues, sometimes little understood or appreciated by outsiders, but the maize geneticist’s style of science devoted to discovery and an unusually strong commitment to cooperation probably contributes to this trend. One of the great practitioners of this style of science was Marcus Rhoades (Figure 1), who often advised beginning graduate students: “Just get in the lab and start to work; you can’t help but find something.” “What are the facts?” was his common refrain to model building and theorizing. Along with his penchant for discovery was a dogged experimentalist attack to explore the parameters and dimensions of a new finding. Rhoades discovered the first case of cytoplasmic male sterility in maize; the independence of the plastid from nuclear control by the action of the iojap mutation; the Dotted “mutator” system that was later found to be a transposable element (see Fedoroff 1998); many features of chromosomal behavior; the “duplicate” nature of the maize genome; effects of supernumerary chromosomes on the normal set; and preferential segregation resulting from neocentromere formation caused by abnormal chromosome 10 (Dempsey 1973, 1983, 1994; Peterson and Peterson 1973). This last topic maintained Rhoades’s interest until the end of his career. It is, in fact, a rather astounding phenomenon: a variant of chromosome 10 will cause blocks of heterochromatin on all chromosomes to proceed to the poles of meiosis ahead of the normal centromeres (Figure 2). For any-one familiar with the usual course of events, the sight of this progression is indeed fascinating. Rhoades was born in Missouri and raised in Kansas (Dempsey 1973, 1983, 1994). Eventually he found his way to the University of Michigan, where he encountered E. G. Anderson “of attached-X fame” (Rhoades’s own phrase). Anderson had just demonstrated, using attached-X females of Drosophila, that crossing over occurred at the four-strand stage of meiosis. Rhoades’s introduction to genetics was a seminar on this topic conducted by Anderson (Dempsey 1994). Rhoades was reportedly befuddled by this topic, but determined to understand it. Under Anderson’s tutelage, Rhoades went on to receive a master’s degree in genetics from Michigan, at which time he was encouraged to enter the Ph.D. program at Cornell to work with R. A. Emerson. Emerson had been Anderson’s own Ph.D. advisor. It was at Cornell that the stellar group of Rhoades, George Beadle, Charles Burnham, Barbara McClintock, and Emerson coalesced to elucidate many features of maize genetics and cytogenetics in general. A photo of this group, together with Beadle’s dog, taken during pollination season has been widely reprinted (e.g., Sheridan 1982; Dempsey 1983; Keller 1983), and many have commented upon the collection of talent that was present at Cornell at that time (see also Nelson 1993). Rhoades showed this picture to his cytogenetics class at Indiana University in 1974 and remarked in an uncharacteristic lapse of modesty, but with characteristic wit, “Even the dog was smart!” It was at Cornell that Rhoades began an enduring friendship with Barbara McClintock (Rhoades 1984). He was a strong advocate of her work and an admirer of her abilities both in terms of cytological technique and interpretation. Rhoades (1984) recalled his role in getting her to apply her cytological skills to the research problems of the Cornell maize genetics group (see Kass and Bonneuil 2003 for a detailed account of all the factors involved). His course material covered all of her studies, and the laboratory instruction included advice handed along from McClintock on how to analyze meiotic chromosome spreads, for example, “to focus up and down through the nucleus to follow the chromosomes along.” It was a rare visit to Rhoades’s lab in which McClintock’s name was not mentioned in the most glowing terms. McClintock shared with Rhoades a penchant for discovery that no doubt contributed to their lasting scientific bond. —Marcus Morton Rhoades in his laboratory at the University of Illinois. (From University of Illinois Bulletin, Vol. 46, No. 63, April 1949. Photo supplied by Ellen Dempsey.) During his time at Cornell, Rhoades made many contributions to maize genetics and wrote his thesis on the first case of cytoplasmic male sterility found in maize. Rhoades regularly recalled his thesis defense. A chemist unfamiliar with the details of genetics was on his committee. After Rhoades was asked to leave the room at the end of the presentation, an innocent set of questions by this outside member to the committee on why cytoplasmic inheritance might be considered unusual led to an extended educational discussion. Time slipped by without the committee realizing that Rhoades was awaiting a decision in the hallway. Friends of Rhoades, seeing him pace back and forth, speculated that he was “in trouble.” As a consequence of this experience, Rhoades always made certain that such discussions in which he took part were kept short. While at Cornell, Rhoades played an important role in the 1932 International Congress of Genetics by preparing a “living chromosome map” in which mutant plants were planted in rows corresponding to their position on the chromosomes (Crow 1992). —Abnormal chromosome 10 and its effect on segregation. (Top) A pachytene smear of abnormal chromosome 10 (on the left). The deeply staining block of heterochromatin near the end of the long arm is unique to this version of the chromosome. The three deeply staining chromomeres proximal to the Ab10 knob are also specific to this chromosome. (Bottom) A smear preparation of anaphase I in microsporocytes homozygous for Ab10. Note the greatly stretched chromatin fibers proceeding to the poles ahead of the normal centromeres. A chromosome pair without knobs is near the center of the image. In this case the homologs are separating normally and do not have extensions toward the poles. (Photos were first published by Rhoades 1952 and were generously supplied by Ellen Dempsey.) The discovery of preferential segregation: From Cornell, Rhoades accepted a position with the United States Department of Agriculture (USDA), first in Iowa and then in Arlington, Virginia. It was in this position that Rhoades discovered the phenomenon of preferential segregation. Albert Longley (1937, 1938) had described an “abnormal chromosome 10” (Ab10) that was present in populations of Native American varieties and in teosinte, the wild progenitor to maize. This chromosome was characterized by Longley as possessing extra blocks of heterochromatin near the end of the long arm of the chromosome. In a simple experiment to test the effect of this chromosome on recombination, Rhoades (1942) crossed a stock carrying Ab10 to a tester line and backcrossed the hybrid. A deviation from a normal 1:1 ratio for the R locus (an anthocyanin pigment marker closely linked to the knob) was observed. Rhoades coined the phrase preferential segregation to describe this phenomenon. In his class he related that his first reaction to seeing those ears was that the off ratio was due to pollen contamination from an unwanted male parent. Then, with an impish smile, he added that his pollination technique could not have been that bad and that there must be a process operating that affected the relative transmission of the two alleles. He was also known to ask, “What famous government building now stands on the field in which preferential segregation was discovered?” Rhoades was an employee of the USDA at the time and his experimental fields were in Arlington. These plots were later abandoned by the USDA and have since become the site of the Pentagon. In the hybrid mentioned above, Ab10 was carrying the recessive allele of r, and the normal chromosome 10 had the dominant allele. A testcross to the recessive r tester line produced an excess of colorless kernels in a 70:30 ratio instead of the expected 50:50 ratio. The altered ratio occurred on the heterozygous ears, but not when the plants were used as a pollen parent. When Rhoades swapped the R alleles so that the dominant marker was linked to Ab10, the majority of kernels were colored. In maize, as in most plants, megasporogenesis produces four cells, but three degenerate and do not develop into ovules. The basal megaspore differentiates into the megagametophyte via a few mitoses to produce the egg, polar nuclei, and associated cells. It was concluded that Ab10 must find its way into the basal megaspore more often than at random. A cytological examination of anaphase in the male flowers of plants with Ab10 (Rhoades and Vilkomerson 1942; Rhoades 1952) showed that parts of the chromosomes other than the centromere proceeded to the poles ahead of the normal spindle attachment sites. Heterochromatic knobs, of which the one on Ab10 is the largest, were good candidates for the regions responsible for this behavior. Albert Longley (1945) tested this proposition using different lines of maize that varied in the position or size of these knobs. Longley had characterized the cytology of many accessions and had a ready knowledge of the appropriate stocks to use. He found that preferential segregation occurred for the knobbed homolog for every one tested, but only in the presence of a copy of Ab10. Thus, any site on a chromosome that has a knob where there is none on the homolog [or a larger version than on the homolog (Kikudome 1959)] will be preferentially segregated to the basal megaspore in the presence of Ab10. As a result, knobs would continue to be selected for greater size when Ab10 is part of the genome. Rhoades’s 1942 article on preferential segregation is an interesting study in objective detachment leading to a logical conclusion. He systematically covers the data that falsify one trivial explanation after another in an asymptotic approach to the conclusion of altered segregation. He even hung plants upside down in the green-house to see if the extra chromatin on Ab10 made it heavier and thus more prone to be present in the basal megaspore (there was no effect). There is a tone of disbelief throughout the article, but having disproved the alternatives, he concluded that preferential segregation must be occurring. The receipt date on the preferential segregation article is listed as December 25, 1941. In 1940 Rhoades had accepted a position in the Department of Botany at Columbia University in New York City. One wonders how an article could possibly be received on Christmas Day? The answer lies in the fact that Rhoades was the Managing Editor of Genetics at the time. The completed manuscript and its submission were apparently a gift to himself. Interestingly, the tone of disbelief in his 1942 article continues even to the Rhoades and Dempsey (1966) article on the topic. One source of concern to Rhoades came from studies of inversion heterozygotes conducted with his long-time research associate Ellen Dempsey (Rhoades and Dempsey 1953). Rhoades had studied Drosophila for a year at Caltech (1929-1930) during his graduate student years under the guidance of Alfred Sturtevant and Theodosius Dobzhansky. He therefore had an appreciation for Drosophila genetics and grasped the implications of the fact that paracentric inversion heterozygotes in Drosophila show little sterility. The basis of this phenomenon is thought to be that the bridge formed by recombination within the inversion loop orients the tetrad of chromatids so that the intact noncrossover chromatid remains in the egg and the abnormal crossover chromatids go to the polar bodies (Sturtevant and Beadle 1936). Rhoades and Dempsey conducted extensive work on inversion heterozygotes in maize and found that a similar orientation of chromatids does not occur. Bridge formation results in broken chromatids that can become included in the basal megaspore at frequencies predicted from random distribution. This dichotomy of results, in that there is an apparent orientation with Ab10 but none with inversion heterozygotes, appeared to plague Rhoades’s thinking on the interpretation for many years. Subsequent studies of megasporogenesis in Ab10 material clearly showed that preferential segregation results from an orientation of knobbed chromatids toward the outer poles of female meiosis (I. Golubovskaya, personal communication). Many of Rhoades and Dempsey’s later studies on Ab10 were included as notes in the Maize Genetics Cooperation Newsletter under some version of the title “Further studies on preferential segregation,” describing work conducted during his tenure at the University of Illinois (1948-1958; Figure 3) and at Indiana University (1958-1974; Figure 4). Rhoades served two terms as the editor of the newsletter, from 1932 to 1935 and again from 1956 to 1974. Ellen Dempsey played a pivotal role in the assembly of the newsletter during the latter period. An offhand comment in Rhoades’s presence about a “little” note in the newsletter was met with the friendly retort that “for Miss Dempsey, the Newsletter is a big publication,” no doubt in honor of her substantial contributions to its yearly production. Rhoades also served on several editorial boards (Dempsey 1973, 1983, 1994). As noted above, he was Managing Editor of Genetics from 1940 until 1948, but continued with service on the editorial board and as an active reviewer of manuscripts until 1966. He served a term as the president of the Genetics Society of America (1943) and of the American Genetics Association (1950-1953). Among the many honors he received, Rhoades shared the first Thomas Hunt Morgan award from the Genetics Society of America in 1981 with Barbara McClintock. —The maize genetics group at Illinois (early 1950s). Front row: Marcus Rhoades, Dwayne Richardson, Mei Lin, S. H. Tulpule. Back row: Ellen Dempsey, John Laughnan, George Ziska, Edward H. Coe. When Rhoades moved to the University of Illinois from Columbia University in 1948, Laughnan was also recruited to the Illinois faculty from Princeton. Dempsey was a research associate with Rhoades. Richardson, Lin, and Tulpule received Ph.D.’s under the direction of Rhoades. Coe was a graduate student with Laughnan. Both Rhoades and Coe were later named recipients of the Thomas Hunt Morgan award from the Genetics Society of America for lifetime contributions to genetics. (Photo courtesy of Susan Gabay-Laughnan.) In his many service activities, Rhoades no doubt made a tremendous impact on the field of genetics. Many wished for his approbation. An attendee at the annual maize genetics meeting once commented, “Rhoades sits in the front row and passes judgment on all the talks.” A more accurate interpretation is that Rhoades had a voracious appetite for maize genetics and high standards as an experimentalist. Rhoades was the perfect gentleman and would break up any heated scientific discussion by commenting, “This is good clean fun, but....” At his last presentation at the maize meetings in 1987 at the age of 83, Rhoades talked on preferential segregation. In his conclusion, he said: “I know this may seem old fashioned to some of you, but it’s the kind of thing that keeps an old maize geneticist like me going.” segregation and meiotic years after Rhoades preferential described an apparently phenomenon in Drosophila caused by a locus named or (see These Rhoades (1942) but not his instead the phrase segregation phrase has Rhoades’s in the as by a of several that found using but only using The phenomenon of meiotic to the in which one member of a pair of homologs is preferentially in the of a a will allele frequencies in a the of the and the by of allele and who first meiotic (see Rhoades’s work on Ab10 as one the of carrying the and meiotic which on the of meiotic At that the model system for the study of meiotic but then it was that heterozygous have a normal and the of and This that the of meiotic be to in production. The case of Ab10, which was discovered remains to the in pollination Marcus Rhoades in the maize at Indiana (Photo courtesy of Ellen Dempsey.) Ab10 and preferential segregation Maize knobs were the first to the of The laboratory of in a that is present in most knobs and on Ab10 These cytological features were found to be of that are present of at any one Rhoades two in the lab and was to the nature of the heterochromatin of Ab10 and the knobs. to from Rhoades and Dempsey in clearly a concern that no one would continue on their chromosome. not have the number of on Ab10 and to their work on preferential segregation have only in the last Rhoades as a of centromere and was in how on the spindle (Rhoades In years, has a of new and interesting details about the of neocentromere in maize. that are active throughout and anaphase and that their of can be as as than normal chromosome of for and the that with fibers instead of as is of centromeres (see Figure This with is probably responsible in part for their of and of the such as centromere and do not to that are centromeres for and have been studied in other as In have to be similar to centromeres and have an important for the and of The term once to cytogenetics has at anaphase with the chromosome by with spindle fibers (Photo by and Rhoades and his also took a strong interest in the associated with Ab10, using the size of the knob Rhoades and Dempsey and many were with to their effects on meiotic neocentromere and another unusual of to recombination throughout the genome. A of new have been in the last as as at two which meiotic but have no cytological and and the data that at four different are for meiotic two more than by Rhoades and A discovery from studies was the that there are two neocentromere on Ab10 data had that a known as with the in many knobs and used in to show that three chromomeres on Ab10 are in by this the have even more than the and often ahead of the knobs in that to the by Rhoades and Dempsey the that are to knobs that a for neocentromere to a proximal of Ab10, and that a for neocentromere to a more site These data that the and the spindle is rather than an of the nature of the in knobs. Ab10 has also the interest of As an of meiotic one must why Ab10 and the knobs it have not been to in maize and under meiotic Ab10 has an of only in those populations in which it and the knobs in the presence of Ab10. the of Ab10 and knob frequencies in Rhoades’s (1942) that Ab10 has a male an as a meiotic and also that knobs in to meiotic by Ab10 that knob and chromosomal position are all effects of Ab10. Rhoades might have the thought that Ab10 was a in the and of the maize genome. is a model for the of to this there is an centromeres for to the more with the and a variant of that with the to be a in the site of the on the chromosome. is to to to centromeres and The in this model are in many on the of meiotic in maize as by Rhoades. could have predicted the of abnormal chromosome 10 and the phenomenon of preferential It a to the a to the over once to the For Rhoades, the of the remains to be about a a and the role of meiotic in the genome. The to these questions will be found by Rhoades’s of his dogged and his and by his advice to “Just get in the lab and start to The John Susan and Ellen Dempsey for their and for her E. E. Dempsey, S. and for on the Ellen Dempsey the of Rhoades and in meiosis. Susan the photo of the Illinois maize genetics