exceptionally high levels of restriction site polymorphism in dna near

Copyright 0 1983 by the Genetics Society of America

EXCEPTIONALLY HIGH LEVELS OF RESTRICTION SITE POLYMORPHISM IN DNA NEAR T H E MAIZE A D H ~ GENE

MITRICK A. JOHNS, JUDITH N. STROMMER' AND MICHAEL FREELING

Departtnent .f Genetics, University of California, Berkeley, California 94720

Manuscript received February 28, 1983 Revised copy accepted August 1, 1983

ABSTRACT

Restriction maps have been prepared for the chromosomal region near seven biochemically and genetically distinct maize alcohol dehydrogenase- 1 (Adhl) alleles using a small cDNA probe for Adhl. Five restriction sites span- ning about 4 kb in and near the Adh l transcription unit appear identical in all seven alleles. Outside this conserved region, variation in restriction site position is the rule. Six of the seven alleles are distinguishable, and the alleles appear to fall into four groups. The DNA flanking the IS-type alleles seems to share no restriction site homology with the DNA near the IF-type alleles. Several hypotheses are put forward to explain how such high levels of polymorphism could have arisen in a species that has been domesticated for only about 10,000 years.

AIZE is a domesticated species: unlike all other food crops, no wild maize M is known, because several morphological and quantitative characteristics make it unfit to survive without human intervention. The origin of maize has been the subject of much debate; currently, two major hypotheses are popular. One theory states that cultivated maize is merely a subspecies of Zea mexicana (teosinte), which has been selectively bred for agronomically useful characteristics (BEADLE 1978; DOEBLEY and ILTIS 1980). The other theory suggests that cultivated maize arose from a now-extinct wild maize, and that teosinte is a more distant relative of maize (MANCELSDORF 1974; RANDOLPH 1976). Today, about 350 morphologically distinct races of maize are recognized (BROWN and GOODMAN 1977), although it is unclear how these races are related phyloge- netically. If all maize races are derived from a single domestication event 10,000 years ago, one might guess that all maize races would have nearly identical DNA sequences in any chromosomal region. Recent work by BURR, EVOLA and BURR (1983) has shown that a great deal of restriction site polymorphism exists near the Shrunken-1 gene and also near several randomly chosen single-copy genes in maize. Here, we confirm and extend these results for the Adhl gene.

The Adhl gene is on the long arm of chromosome 1, about 80-90% of the distance from the centromere to the telomere (BIRCHLER 1980), at map position 127 (see FREELINC and BIRCHLER 1981). Several naturally occurring var- ' Current address: Department of Genetics, University of Georgia, Athens, Georgia 30602.

Genetics 105: 733-743 November, 1983.

734 M. A. JOHNS, J. N. STROMMER AND M. FREELING

iants of Adhl are known (see FREELING and BIRCHLER 1981). Because a cDNA probe to the 3’ half of the ADHl mRNA was available (GERLACH et al. 1982), and because Adhl hybridizes as a unique sequence at Tm-20” (STROMMER et al. 1982), we were able to prepare genomic restriction maps covering about 20 kb of DNA on the chromosome surrounding Adhl . The maps of seven Adhl chromosomal regions using seven restriction enzymes are reported here.

The seven lines of maize were not chosen at random; rather, they carry Adhl alleles previously studied and of continuing inerest to us. The alleles vary in their electrophoretic behavior, specific enzyme activity, amount and quality of intragenic recombination or quantitative, organ-specific expression. The origins and characteristics of these seven alleles are detailed later in this paper. Three alleles are from Corn Belt maize, one allele has been recently intro- gressed from teosinte and the other three alleles probably originate from different Latin American races. Our sampling of the extant maize races is thus very limited.

We have found a large amount of variation among the Adhl alleles studied. We suggest several possible explanations for this variability: (1) Our selection of interesting alleles may mean that we studied only the unusual, extreme variations within maize. (2) The sequences near Adhl may be composed of highly mobile elements, similar to the middle repetitive sequences in Droso- phila, most of which are thought to be mobile over evolutionary time (YOUNG 198 1). (3) Corn may have undergone multiple, independent introgressions from teosinte, which may have evolved many different Adhl alleles over a long evolutionary history. (4) The process of domestication may have created or selected “genetic catastrophes” involving major rearrangements of noncoding DNA sequences (see, for example, GOLDSCHMIDT 195 1).

MATERIALS AND METHODS

Molecular methods: DNA was extracted from the leaves of mature plants using the method of MURRAY and THOMPSON (1980), followed by two cycles of CsCl density gradient centrifugation in 4.5 M CsCl and 200 pg/ml of ethidium bromide. The ethidium bromide was removed by butanol extraction, and the DNA was dialyzed against 10 mM Tris-HCI, 1 m M EDTA, pH 7.5. Aliquots of 15 pg of DNA were digested with 40 units of restriction endonuclease (from Bethesda Research Laboratories) for 2 hr. Digestions were performed in a buffer containing 10 m M Tris HCI, pH 7.5, 10 m M MgC12, 5.0 m M &mercaptoethanol and 0.1 mg/ml of sterile gelatin. The buffer for BglII, Hind111 and BstEII also contained 50 m M NaCI, and the buffer for BamHI, SstI and XbaI contained 100 m M NaCI. The buffer for KpnI contained no added NaCI. All digestions were performed at 37”, except that BstEII digestions, which were carried out at 60”. Double digestions were performed sequentially, with appropriate adjustment of the ionic strength and temperature. After digestion, the DNA was precipitated with ethanol, then resuspended in electrophoresis buffer (89 m M Tris, 89 m M boric acid, 3 m M EDTA). Agarose gels of 0.7% were run at 2.0 V/cm for 17 hr, and then the DNA was nicked by soaking the gel in 1% HCI for 15 min. The gels were then denatured in 0.5 N NaOH, 1.5 M NaCl for 30 min, neutralized in 0.5 M Tris HCI, pH 7.0, 3.0 M NaCl for 60 min and blotted onto nitrocellulose filters according to the method of SOUTHERN (1975), using 10 X SSC as a transfer buffer. The filters were dried under vacuum at 75” and then hybridized in Denhardt’s solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine Serum albumin) containing 1.0 M NaCI, 0.05 M sodium phosphate, pH 7.4, 1 m M EDTA, 0.2% SDS and 0.15 mg/ml of denatured salmon sperm DNA. Included in this solution was 0.2-0.5 pg of Adhl- cDNA, isolated from the plasmid pZML84 as an 880-bp PstI fragment (GERLACH et al. 1982), and

MAIZE RESTRICTION SITE VARIATION 735

labeled with by nick translation (RIGBY et al. 1977) to a specific radioactivity of about 10’ cpm/cg. Hybridization was carried out at 65” for 40 hr, after which the filters were washed in three changes of 0.1 SSC, 0.1% SDS at 65” for a total of 1 hr. The filters were then exposed to Kodak XAR-5 film backed with a Dupont Lighting Plus intensifying screen at -70” for 1-7 days. Only the fragments containing Adhl sequences showed significant hybridization-Adh2 was not detected. Migration distances were converted to kilobase pairs by comparison of the bands hybrid- izing to the cDNA probe with a standard of bacteriophage X wt DNA that was digested with HitidIII and run on each gel.

Mapping strategy: Genomic DNA cut with a restriction enzyme consists of a large number of fragments of different lengths which can be separated by electrophoresis; the fragments homologous to the cDNA probe are detected by hybridization with a labeled probe. The region of genomic DNA homologous to the probe is about 1.5 kb long, and it consists of several exons separated by introns. Of the seven enzymes we used for mapping, only Hind111 cuts within the region homologous to the cDNA probe. Thus, for each of the other enzymes, a single fragment of genomic DNA hybridized with the probe; this fragment contains the region homologous to the probe, and it is bounded by one restriction site 5’ to the probe and another site 3’ to the probe (see Figure 2). For HindII1, there is also a central restriction site within the region homologous to the probe; for this reason, two fragments of HindIIIdigested genomic DNA hybridize with the probe.

As shown in Figure 1, all seven of the alleles that we mapped had a common 2.5-kb HindIlI fragment. STROMMER et al. (1982) showed that this fragment extends 5’ from the central HindIII site in Adhl-IS, and it was reasonable to assume that the central Hind111 site and the 5’ site were in the same position in all of the alleles. The other HindIII fragment shown in Figure 1 is the 3‘ fragment; its size gives the distance from the central HindIlI site to the 3’ site. Thus, the data in Figure 1 allow the derivation of a simple map for each allele, containing the region homologous to the probe, the central Hind111 site and the 5’ and 3’ flanking Hind111 sites.

If a second restriction enzyme cuts within either of the HindIII fragments, a comparison of genomic DNA digested by both Hind111 and the second enzyme with genomic DNA cut by HindIII alone will show that one of the fragments in the double digest is shorter than the corresponding Hind111 fragment. The size of this shorter fragment gives the distance from the central Hind111 site to the site for the second enzyme. Digestion by the second enzyme alone gives a fragment whose length is the distance from the previously located site to the unlocated site on the other side of the probe. Sites for enzymes not cutting within the HindIII fragments can then be determined using their overlap with fragments already mapped with respect to the HindIII sites. In this fashion, a consistent and unique map is constructed. The apparent lengths of restriction fragments varied 5-10% between gels; thus, it was necessary to resolve the order of closely spaced sites by running side-by-side comparisons of appropriately digested DNA.

Genetic material: The Adhl-IS and Adhl-IF alleles (abbreviated IS and I F , respectively) are the original alleles used by SCHWARTZ (1966) to define the Adhl structural gene genetically. They were isolated from a single heterozygous seed of an American Corn Belt hybrid line, and we are sure that all of the IS seed used in this lab is derived from a single alelle. Some confusion concerning the ancestry of IF exists; it is possible that two rather than one allele are currently called ‘ I F.” The original difference noted between these alleles is that during electrophoresis the product of the IS allele migrates more slowly toward the anode than does the product of the I F allele. SCHWARTZ (1971) noted that the IS and I F alleles are expressed differently in different organs. In the scutellum (an embryonic storage organ), IS and IF are expressed approximately equally, but in the anaerobic primary root, IF makes twice as many ADH subunits as does IS. SCHWARTZ proposed an elegant model, involving the competition between alleles for a limiting factor, to explain these observations (SCHWARTZ 1971; JOHNS, ALLEMAN and FREELINC 1983).

FkF is found in the commercial hybrid line Funk G4343. Because this is not an inbred line, two different Adhl alleles may be present. However, we have not been able to discern any differences between these two putative alleles by electrophoretic comparison of isozymes or by restriction site mapping. Nevertheless, it is possible that mapping at a finer scale will distinguish two different Adhl alleles in the G4343 line.

FREELING (1978) studied the intragenic recombination between null mutants induced in the IS,

736 M. A. JOHNS, J. N. STROMMER AND M. FREELINC

CmCt IS 54s IF FkF 33F

: - m a - I- -

-Origin

-23.7 kb

-9.5

- 6.6

-4.5

- 2.0

FIGURE I.-An autoradiograph of a blot hybridization of HindIIkut genomic DNA from our seven genotypes, hybridized with radiolabeled pZML84. a cDNA for the 3' half of the ADHl messenger RNA. The 5' fragment common to all alleles migrates at a position corresponding to about 2.5 kb; in all lines except Cf. the 3' fragment is larger. The positions of the origin and the size standards are shown.

IF and FkF alleles by scoring stained pollen grains for normal levels of enzyme activity, whose rare appearance resulted from the intragenic recombination of two different null alleles to produce a functional Adhl gene. He found that the parental allele strongly affected the frequency of recombination. Different null alleles induced in IS recombined with each other at a frequency nine times the frequency that null alleles induced in IF recombined among themselves. However, IS null alleles did not recombine with IF null alleles at all. Interestingly, a null allele induced in FkF recombined with both IS and IF alleles.

Cm is an allele from a Colombian cultivar whose product migrates faster than the IF product. Cm specifies subunits that have '/w the enzyme activity per molecule as IF subunits, although the Cm and IF alleles produce approximately equal numbers of molecules (&WAR= and LAUGHNER 1969). The Ctn allele does not recombine with IF, IS or FkF (FREELING 1978).

Cf makes a product of normal enzyme activity per molecule which migrates to the same 'extra- fast" position as the Ctn product. The Adhl allele in Cf was derived from a teosinte line (Z. mexicam), by crossing maize and teosinte, then repeatedly backcrossing to maize and selecting for the teosinte Adhl allele (SCHWARIZ and ENDO 1966).

The 33F gene product has the same electrophoretic mobility as the IF product; this allele is derived from the Super Gold Popcorn line. WOODMAN and FREELING (1981) found that. compared with IF, 33F is relatively underexpressed in the root and overexpressed in the scutellum.

54s makes a product that migrates during electrophoresis like the IS product. 54s is derived from a line originally described by EFRON (1971), which was thought to carry a regulatory locus that acted in f r m s to decrease the amount of ADH made in the seed and which mapped about 17 cM from Adhl. WOODMAN and FREELING (1981) found that this line contained an element


near or in Adh l which acted in cis to cause overexpression in the anaerobic root and underexpres- sion in the scutellum, compared with IS. WOODMAN and FREELINC did not find a trans effect in progeny from EFRON’S line (unpublished data).

33F and 54s represent two of the alleles most different from IF and IS among the alleles WOODMAN and FREELING studied. However, all of the alleles conformed to the “reciprocal effect” rule: any allele that is overexpressed in one tissue is underexpressed to the same extent in the other tissue (see discussion in JOHNS, ALLEMAN and FREELINC 1983).

RESULTS

Figure 2 illustrates the restriction maps of the seven Adhl alleles that we have studied. Several unusual features of the maps need to be explained. (1) We found that digestion at the 3‘ BamHI site in IF and FkF was usually not complete, and a second BamHI site further 3’ could be mapped; thus, two BamHI sites 3’ to the probe are located on the maps of these alleles. (2) Some digestions, such as the KpnI digestion of 54S, produced fragments of greater than 20 kb in length. Restriction sites cannot be accurately mapped from such data, so they are marked on the maps with wavy lines to indicate ambiguity of position. In several cases, the order of two outlying sites could not be determined; these sites are located together in parentheses. (3) The mapping method described worked for all alleles except Ct. For this allele, only the HindIII, BgZII and XbaI fragments showed partial overlap. Outside this area, the BamHI fragment was completely contained within the SstI fragment, which in turn was completely contained within the KpnI fragment. The BstEII sites are apparently very close to the KpnI sites, since the BstEII-KpnI double digestion fragment is almost exactly as long as the BstEII and KpnI single digestion fragments. Thus, the relative positions of the BamH1, SstI, KpnI and BstEII sites are known, but the distances between them and the distances to the central HindIII site are unknown. This fact is denoted by wavy lines separating these sites in Ct.

The maps in Figure 2 clearly demonstrate two things: within and adjacent to the Adhl transcribed region the restriction sites are identical in all seven chromosomes, and outside of this conserved region there is a great deal of variability.

The conserved region consists of about 4 kb of DNA, demarcated by the 5’ sites for BamHI, XbaI, HindIII and BgZII, and the central HindIII site. Tran- scription begins between the 5’ XbaI and HindIII sites (E. DENNIS and W. J. PEACOCK, personal communication), and the poly-A addition site lies about 1,000 bp 3’ to the central HindIII site. Thus, the conserved region consists of the transcription unit and at least 1 kb of DNA 5’ to the start transcription.

Although our mapping detected no differences in the coding regions of these alleles, other work has demonstrated that the amino acid-coding portion of this region varies among the alleles, in that three electrophoretic variants are represented among these seven alleles, and Cm makes a product that has a lower specific enzyme activity per molecule than the product of IF (FREELING and BIRCHLER 1981). Thus, the transcription unit of Adhl can only be described as relatively conserved, not completely conserved. Restriction mapping


Restriction Si te Variation at Adh 1

Cm

s I x i n o n \ x I E I n 0 U ! I I IO I I I l l I I 1 1 I I

I

I I I I

I Ct

(UsEl S B x ; n 0 n x n 0 I S 1U.E) I 1 . 1 I 1 , ) 1 I I ; ( I , I 1 I

I

54s

I

I

1F (U.El S E x i n Q U Q SE

I . 1 1 : ) I l I I II I I 1 I

I

I

FkF 1U.EI s E X : P C H 0 S I X U U (B) E

I 1 1 , l I I , 1 I 1 I I , I I 1

I

33 F I

K E X : H G n ~ 8 X E n O E U l l I , I 1

E S , I I

I 1 I 1 I ; I I 1 1 t

H I i 6.n H I

E c 0.1 E l l I( z Upn I 1 Lb

0 E I q l I 1 s = S.1 I

n I nand III X = X b a I

FIGURE 2.-Restriction maps of the seven Adhl chromosomal regions. The maps were prepared as described in the text. The boundaries of the transcription units are denoted by the vertical dashed lines, and the region that hybridized with the ADHI-cDNA probe, pZML84, is also shown. Several outlying sites are separated from the main body of certain maps by wavy lines; these indicate that the exact position of the outlying sites are unknown, either because the fragments bounded by these sites were too long to be accurately measured or, in the case of Ct, because no overlapping fragments were found that allowed positioning with respect to the central Hind111 site. The symbols for some of the outlying sites are grouped in parentheses, indicating that their order is uncertain. A fuller discussion of mapping is presented in the text.


is not capable of detecting most of the small scale genetic changes that could alter enzyme-specific activity or electrophoretic mobility.

Outside the conserved region, restriction site polymorphism is the rule. Ex- amination of the maps in Figure 2, especially the regions 3’ to the transcription units, shows that the seven alleles can be grouped into four categories based on apparent similarity: ( I F and FkF), ( I S and 54S), ( 3 3 F and Cm) and Ct. IF and FkF are indistinguishable by the restriction mapping we have done. I S and 54s are very similar in the 3’ region, showing only a single polymorphism for the 3’ SstI site. On the other hand, the 5’ regions of the two alleles show no similarities. In the 3’ region, 33F and Cm share four restriction sites (SstI, BstEII, Hind111 and BglII), while differing in the other three sites. The regions 5’ to the transcription units in Cm and 33F show no apparent homology. Ct , an Adhl allele from teosinte, appears quite different from the other alleles.

Outside the transcribed region, IS and IF apparently share only the 3’ Xbal site. In general, the nonconserved areas of IS and IF look like completely different sequences of DNA. We see three possible- explanations for the differences between IF and IS. (1) These two alleles could be basically similar in sequence, except for a (presumably large) number of base change mutations which have altered the restriction sites. (2) Several small insertional mutations could have occurred, with some of the inserts containing sites for the enzymes we used. (3) The DNA 3’ to the transcription unit could be completely different between IS and I F , the result of a large insertion or inversion. It is important to note that examination of paired pachytene chromosomes of I S / 1 F heterozygotes revealed no gross chromosomal rearrangements in the neigh- borhood of Adhl (FREELING 1978). We are unable to choose among these alternatives, but we favor the third possibility, because it could occur in a single step, consistent with the relatively short time that maize has existed as a species.

We conclude that the 20-kb region around Adhl contains at least two classes of sequences: highly conserved DNA that includes the Adhl gene and variable DNA in the flanking regions. Clearly, these two classes of DNA have evolved, naturally or artificially, at vastly different rates.

DISCUSSION

The relationship between restriction maps and recombination frequencies: As dis- cussed in MATERIALS AND METHODS, null alleles induced in I F and I S do not recombine with each other, although a null allele induced in FkF recombines with both IS and IF null alleles (FREELING 1978). From these observations, we can make some predictions concerning how similar the sequences flanking different alleles should be. We expected that IF and IS should be quite dissim- ilar, so as to inhibit effective pairing, whereas FkF should share some similarities with both alleles. The restriction maps show that I F and IS are indeed very different, except near the transcription unit; possibly, pairing is inhibited in this region. However, the FkF allele is indistinguishable from IF using restriction maps, and it can recombine with either I F or IS. It is possible that


the recombination of IS and FkF is due to a "marker effect," since only one null alllele induced in FkF was studied. However, the restriction map of this null allele is not different from that of the parental FkF allele ( M . A. JOHNS, J. N. STROMMER and M . FREELING, unpublished data), and no allele of 1 s and I F deviates from the rule that 1 s and 1F alleles do not recombine. Apparently, the crucial differences between these alleles have not been detected by our restrictional mapping.

Polymorphism at A d h l compared with other loci: The A d h l locus in maize appears very polymorphic when restriction sites are compared. In a similar study, BURR, EVOLA and BURR (1983) were able to distinguish four alleles of S h l among six common American Corn Belt inbred lines using two restriction enzymes. They further determined that nine of 16 random cDNA clones that hybridized to unique loci could detect polymorphism among these six inbred lines when digested by a single restriction enzyme. It is clear that A d h l is not unique in maize in being highly polymorphic.

Patterns of restriction site variation similar to the apparent base substitutions between IS and 54S, and between 33F and Cm, have been found at the Dro- sophila Adh gene (LANGLEY, MONTGOMERY and QUATTLEBAUM 1982) and the 87A and 87C heat shock loci (LEIGH BROWN and ISH-HOROWICZ 1981) among the species of the melanogaster subgroup. The heat shock loci data are perhaps the most directly comparable to ours, due to similar mapping strategies. A number of sites were mapped both within the transcribed region and adjacent to this region, and it was found that, within the transcription unit, there was very little variation between D. melanogaster, D. simulans and D. mauritaana, but much variation was discovered outside this region: 13 of the 58 sites studied differed among these three species.

LANGLEY, MONTGOMERY and QUATTLEBAUM (1982) studied the Adh locus among these same Drosophila species and others and also found large amounts of variation outside the transcription unit, most of which appeared to be due to base pair changes. They found very few polymorphic sites among different isolates of D. melanogaster (only four sites of 24 showed any polymorphism among the 18 lines examined), although a number of insertional changes were detected. When our maps are compared with the Drosophila Adh and heat shock maps, it appears that there is as much variability among different lines of corn as had been found among Drosophila species that have been separate species for millions of years.

This observation is especially evident when IS and I F are compared. These alleles were both found in commercial Corn Belt corn, and yet their restriction maps are completely different, sharing only one site among the ten studied outside the transcribed region. Differences this drastic do not exist among the three most closely related Drosophila species, D. melanogaster, D. simulans and D. mauritiana for either of the two loci studied. A comparable level of variations can be found only when one compares the Adh alleles of such distantly related species as D. melanogaster and D. yakuba, which share at most eight sites among the 24 mapped outside the transcription unit.

Restriction site polymorphisms have also been identified at the human @-


globin genes (JEFFREYS 1979) and the insulin gene (BELL, KARAM and RUTTER 1981), but the amount of variability found was slight. A highly polymorphic locus of unknown function has been detected among humans (WYMAN and WHITE 1980), but in general, humans seem to have little variation in restriction sites compared to maize.

Possible origins of restriction site variation near Adhl: It is, of course, consid- erably easier to find a case of exceptional sequence variation than it is to explain it. We suggest four possibilities.

1. The seven chromosomal regions compared were not chosen at random but rather because they contained clearly different Adhl alleles. These alleles are described in MATERIALS AND METHODS; each is uniquely defined by some combination of ADH 1 surface charge, intragenic recombination behavior or organ-specific quantitative expression. It is possible that the restriction site polymorphisms we have discovered are somehow related to these regulatory and recombinational differences, but no clear pattern emerges when the restriction maps are compared with the phenotypes. It will be interesting to prepare restriction maps from races of maize chosen solely on the basis of geographical separation. 2. It is possible that maize Adhl is a small, approximately 4-kb island in a

sea of mobile, middle-repeat DNA. Except for the genes encoding the stable RNAs, middle-repeat DNA in Drosophila has been shown to consist largely of mobile, copia-like sequences ($ YOUNG 1981). HAKE and WALBOT (1980) es- timated that 40% of the maize genomic DNA was middle-repeat sequences

= 0.3-70). Perhaps, as in Drosophila, the huge middle-repeat compo- nent of maize DNA is mobile.

3. Perhaps the high level of restriction site polymorphism reflects an equally large amount of variation among the progenitors of maize. If maize was domesticated several times independently ($ GALINAT 1977), or if the gene pool incorporated into maize at the time of its speciation was large and variable, then the allelic variation we have discovered is really not 10,000 years old but as old as teosinte or the genus Zea.

4. The least conventional possibility invokes the process of domestication itself as being linked, indirectly, with restriction site polymorphism. MANGELS- DORF (1947) suggested that, if teosinte were indeed the progenitor of maize, then the speciation process must have involved “cataclysmic changes” in the genome. Such an event could also provide the major morphological and restriction site differences seen between the races of maize. We suggest that recurrent, artificial selection for agronomically useful traits, coupled with partial protection by humans from environmental stresses, might have selected for rare genomic shufflings; perhaps, the middle-repeat DNAs move around en masse occasionally, either as a random event or in response to certain stimuli. Thus, domesticated species might display more sequence variation outside of genes than their wild relatives. This possibility clearly fits GOLDSCHMIDT’S (1951) “hopeful monsters” description of the kinds of mutants that underlie speciation. Comparisons of genomic variability between domesticated and wild species should help test this hypothesis.


This research was supported by National Institutes of Health grants GM28600 (to M. F. and W. C. TAYLOR) and GM21734 (to M. F.). M. A. J. and J. N. S. are both National Institutes of Health Postdoctoral Fellows.

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