CHLOROPLAST DNA VARIATION IN PEARL MILLET AND RELATED SPECIES · various regions of the pearl millet chloroplast genome were used as hybridization probes. The first recombinant DNA

Copyright 0 1984 by the Genetics Society of America

CHLOROPLAST DNA VARIATION IN PEARL MILLET AND RELATED SPECIES

MICHAEL T. CLEGG,**+ JAMES R. Y. RAWSON* AND KAREN THOMAS*

*Departments .f Botany and tMoleculur and Population Genetics, University of Georgza, Athens, Georgza 30602

Manuscript received August 2, 1983 Revised copy accepted November 16, 1983

ABSTRACT

The evolution of specific regions of the chloroplast genome was studied in five grass species in the genus Pennisetum, including pearl millet, and one species from a related genus (Cenchrus). Three different regions of the chloroplast DNA were investigated. The first region included a 12-kilobase pair (kbp) EcoRI fragment containing the 23S, 16s and 5 s ribosomal RNA genes, which is part of a larger duplicated region of reverse orientation. The second region was contained in a 21-kbp Sal1 fragment, which spans the short single-copy sequence separating the two reverse repeat structures and which overlaps the duplicated copies of the 12-kbp EcoRI fragment. The third region was a 6-kbp EcoRl fragment located in the large single-copy region of the chloroplast genome. Together these regions account for slightly less than 25% of the chloroplast genome. Each of these DNA fragments was cloned and used as hybridization probes to determine the distribution of homologous DNA fragments generated by various restriction endonuclease digests.-A survey of 1 2 geo- graphically diverse collections of pearl millet showed no indication of chloroplast DNA sequence polymorphism, despite moderate levels of nuclear-encoded enzyme polymorphism. Interspecific and intergeneric differences were found for restriction endonuclease sites in both the small and the large single-copy regions of the chloroplast genome. The reverse repeat structure showed identical restriction site distributions in all materials surveyed. These results suggest that the reverse repeat region is differentially conserved during the evolution of the chloroplast genome.

ECENT advances in molecular biology provide a more detailed view of R evolutionary processes. T w o types of data are beginning to accumulate. The first derives from comparative analyses of complete DNA sequences (MONTGOMERY et al. 1980; BROWN, PRAGER and WILSON 1982; AQUADRO and GREENBURC 1983; BROWN and CLEGG 1983; ZURAWSKI, CLEGG and BROWN 1984). The second class of data comes from the use of type I1 restriction endonucleases to fragment DNA molecules at specific recognition sites. Be- cause restriction endonucleases cleave DNA molecules if, and only if, a specific permutation of nucleotides occurs (usually four, five or six nucleotides in length), the DNA sequences at the termini of the cleavage products are pre- cisely defined. Restriction endonucleases can thus be used to detect genetic differences among individuals in a population, because nucleotide substitutions

Genetics 106: 449-461 March, 1984.

450 M. T. CLEGG, J. R. Y. RAWSON AND K . THOMAS

that create, or destroy, restriction sites will alter the pattern of cleavage products. Moreover, events that lead to the addition, loss or rearrangement of DNA sequences can also be detected (WYMAN and WHITE 1980).

The utility of restriction analysis for the study of genetic variation has been firmly established. The most extensive surveys have concentrated on the DNA sequences coding for the P-globin gene family in human populations (KAZAZIAN et al. 1983) and on the mitochondrial genome from a variety of mammalian species (AVISE, LANSMAN and SHADE 1979; FERRIS, WILSON and BROWN 1981). These studies show a high level of nucleotide sequence variation; they also show that the pattern of fragment change contains information on the sequence of mutational events that separate different lineages.

Animal mitochondrial DNAs (mtDNA) have proved to be particularly useful for the study of sequence variation because these genomes are small [approximately 16 kilobase pairs (kbp)]. Digestion of mtDNA molecules with a typical six-base restriction endonuclease will yield from three to six fragments that can be easily separated by gel electrophoresis. In principle, the rate of nucleotide substitution can be estimated from the fraction of fragments that comi- grates among pairs of different individuals by a procedure referred to as the “fragment method” (UPHOLT 1977; NEI and LI 1979). Estimates of rates of nucleotide substitution for mammalian mtDNAs show a much more rapid rate of evolution than estimated for nuclear single-copy DNA (BROWN, PRAGER and WILSON 1982).

Studies of plant mtDNA variation are more limited, and the data that do exist are more difficult to interpret because of the greater complexity of plant mtDNA (LEVINGS 1983). In addition, there is some evidence for molecular heterogeneity among mtDNA molecules within individuals (DALE 198 1). SED- ERHOFF et al. (1981) and TIMOTHY et al. (1979) have reported mtDNA variation among different lineages of maize and teosinte. Much of this variation has been shown to arise from extensive rearrangements of mtDNA sequences, perhaps due to recombination among different mtDNA molecules (SEDERHOFF et al. 1981). Consequently, the mode of mtDNA evolution in plants differs from the mode observed in animals.

Sequence variation has also been reported among chloroplast DNA molecules (cpDNA) in several groups of plants including Zea, Nicotiana, Lycoper- sicon, Brassica, Triticum, Aegilops, Oenothera and Hodeum species (TIMOTHY et al. 1979; SCOWCROFT 1979; KUNG, ZHU and SHEN 1982; PALMER et al. 1983; BOWMAN, BONNARD and DYER 1983; GORDON et al. 1982; PALMER and ZAMIR 1982; CLEGG, BROWN and WHITFELD 1984). The chloroplast genome is approximately 130 to 150 kbp in size, so that 25 fragments would be expected following digestion with many six-base restriction endonucleases. Unambiguous separation of so large a number of fragments is not possible, and it is difficult, therefore, to employ the fragment method to estimate rates of nucleotide substitution. For this reason, and because we wished to investigate the distribution of variant restriction sites with respect to genome function and structure, we have studied genetic variation using cloned cpDNA sequences. We have investigated several species within the genus Pennisetum including the

CHLOROPLAST DNA VARIATION 451

grain crop pearl millet (P. americanum), as well as one species from the related genus Cenchrus. The resulting data show that certain cpDNA sequences are highly conserved, whereas other sequences show variation at or beyond the interspecific level.

MATERIALS AND METHODS

Plant collections: The grass species used in this investigation are listed in Table 1. In addition to the species listed, 12 samples from within P. americanum were also included in all comparisons. One of these 12 samples is Tift 23DB, an agronomic variety in wide use in India and the south- eastern United States. The remaining 11 samples were obtained from the United States Depart- ment of Agriculture (USDA) World Collection of pearl millet. These 11 samples span the mor- phological and geographic diversity of pearl millet and include entries from India and the range of Subsaharan Africa.

DNA ~nethods: Procedures for the purification of chloroplast DNA and total plant DNA (nuclear, chloroplast and probably mitochondrial) from single-plant samples are given in RAWSON et al. (1981) and RAWSON, THOMAS and CLECC (1982). Three recombinant DNA molecules containing various regions of the pearl millet chloroplast genome were used as hybridization probes. The first recombinant DNA molecule, Ch9. M (cp.rDNA), contains a 12-kbp EcoRI fragment carrying the 23S, 16s and 5 s ribosomal RNA genes. The second cloned molecule was the plasmid pMCS1, which contained a 21-kbp Sal1 fragment. The third was ChS.M(pECPR1) and carried a 6-kbp EcoRI fragment. The cloning and restriction endonuclease mapping of these cpDNA sequences are described in RAWSON et al. (1981).

Chloroplast and total DNA preparations were limit digested with individual restriction endonucleases and separated on agarose gels of varying concentration (0.8, 1.0, 1.5 and 2.0% agarose). DNA was denatured and eluted from the agarose gels onto strips of nitrocellulose filter paper (Millipore HA, 0.45 rm) according to the method of SOUTHERN (1975). The Southern imprints were hybridized with 82P-labeled DNA that contained cloned pearl millet cpDNA sequences. Hy- bridization conditions and procedures used to nick-translate DNA samples are given in RAWSON et al. (1981). Comparison of autoradiographs prepared from total DNA digests and from cpDNA digests verified that the fragments homologous to each cloned DNA sequence employed in this study are located on the chloroplast genome. One microgram of XDNA, limit digested with HindIII, was included on each gel as a molecular size standard.

Isozyme procedures: Eight- to 10-day-old seedlings were extracted in 50 PI of a solution containing 7.6 mM KfHP04, 30 mM cysteine, 1 mM EDTA, 0.40 M sucrose, 0.20 M Tris, 15 mM sodium citrate and 6% (w/v) polyvinylpyrrolidone (pH 7.5). In order to induce alcohol dehydrogenase isozymes, plants were maintained under flooded conditions for 2 days prior to extraction. Root, leaf and stem tissues were combined in all assays.

The enzymes assayed included isocitrate dehydrogenase (IDH), 6-phosphogluconate dehydrogenase (GPGD), phosphoglucoisomerase (PGI), leucine aminopeptidase (LAP), glutamic-oxaloacetic transaminase (GOT), glutamate dehydrogenase (GDH), phosphoglucomutase (PGM), malic dehydrogenase (MDH), alcohol dehydrogenase (ADH) and esterase (EST). Staining solutions are described by BREWER (1970).

Gels of 11% Electrostarch were run for 5 hr at a constant current of 40 mA at 4" using the following bridge and gel buffer systems: 0.41 M sodium citrate, pH 7.0 (bridge), and 5 mM histidine, pH 7.0 (gel), for IDH, GPGD, GDH, PGM, and MDH and 28 mM LiOH with 0.19 M boric acid (bridge) and 4 mM citric acid, 46 mM Tris, 3 mM LiOH and 22 mM boric acid (gel) for GOT, PGI, LAP, ADH and EST.

RESULTS

Chloroplnst DNA restriction fragment analyes: A diagram of the pearl millet chloroplast genome is given in Figure 1, together with the map location of the three cloned millet cpDNA sequences used as hybridization probes. The clone

452 M. T. CLEGG, J. R. Y. RAWSON AND K. THOMAS

TABLE 1

Plant species investigated

Species Abbreviation“ Chromosome

no.

P . a inericn n u in P . jlacciduin P . orientale P. p u rpu rpeuin P . setaceuin C. setiFerus

a f

P

CS

0

S

2 X b = 14 4X = 36 4X = 36 4 x = 28 3 X = 27 4X = 36

“These abbreviations are used in all subsequent tables. X refers to haploid chromosome number.

CI

FIGURE I.-Schematic diagram of the pearl millet chloroplast genome. The locations of the cpDNA sequences contained in the hybridization probes Chg.M(cp.rDNA), pMCSl and Chg. M(pECPR1) are shown. The reverse repeat regions are represented by the heavy line.

Ch9. M(cp. rDNA), includes the 1 2-kbp EcoRI cpDNA fragment coding for the 23S, 16s and 5s rRNA and is located entirely within a large (approximately 22 kbp) reverse repeat structure (RAWSON et al. 1981), which is characteristic of most higher plant chloroplast genomes (BEDBROOK and KOLODNER 1975).


Thus, the DNA sequences included within the Ch9. M(cp.rDNA) probe exist twice on the chloroplast genome. The cloned millet cpDNA sequences contained on the plasmid pMCSl overlap the two identical inverted repeat regions and include 12 kbp of single-copy DNA, which separates the repeat structures. Detailed restriction maps of pMCS1 and Ch9.M(cp.rDNA) have been pub- lished (RAWSON et al. 1981) and are outlined in Figure 2. The map location can be used to predict the expected number of restriction endonuclease sites and the expected number of DNA fragments generated by a given digest. (The EcoRI sites in the small single-copy region spanned by pMCS1 produce many small fragments and have not been mapped.) It is thus possible to un- ambiguously recognize restriction endonuclease site changes within one of the two duplicate regions. The third hybridization probe, Ch9. M(pECPR l), contains a 6-kbp EcoRI DNA fragment of the millet chloroplast genome and maps in the large single-copy region of the chloroplast genome as shown in Figure 1.

Variation among sequeizces homologous to C h 9 .M(cp. rDNA): Our survey of nucleotide sequence variation using the probe Ch9. M(cp. rDNA) gave identical fragment patterns for all 12 lines of pearl millet and all five related species. A total of three four-base and seven six-base restriction endonucleases was used in the survey (Table 2). We can use the restriction map generated with the six-base enzymes to count the number of nucleotides observed within these duplicate regions (282 nucleotides). Although the restriction endonuclease sites for the four-base enzymes have not been mapped, we estimate that a total of 176 nucleotides was detected using this class of enzymes (assuming that each observed band represents two identical fragments). Thus, a total of approxi-

Sal1 I I I L

BamH1 I I I I 1 1 I 1 1

Pst I I I I I I I I

Xhol I I I I I I 1 I

55 235

55

FIGURE 2.-A restriction map of the cp.rDNAs and the short single-copy DNA sequence of pearl millet. The cp.rDNA of pearl millet consists of two inverted repeats, each containing a complete rRNA gene cluster. The two rDNA sequences are separated by 12 kbp of single-copy DNA. This region of the cp.DNA of pearl millet is represented by two recombinant DNA molecules: Chg.M(ct.rDNA) and pMCS1. The EcoRI sites in pMCSl were numerous and thus not mapped.


TABLE 2

Restriction endonucleases used to digest DIVA preparations

Enzyme

EcoRI Bn iii H I Hind111 PstI Sal1 Xhol HpoI HpaII HwIII RsaI

Recognition site pMCSl Ch9.

M(pECPR1)

GAATTC GGATCC AAGCTT CTGCAG GTCGAC CTCGAG GTTAAC CCGG GGCC GTAC

X X X

X X

X

X X X X

X

The probes used with each digest are indicated by an x.

mately 458 nucleotides was included in the recognition sites of the enzymes used in this study. (In making this calculation, we have assumed that none of the four-base sites overlaps a six-base site.)

An estimate of the maximum fraction of nucleotide substitutions can be made using the following assumptions: First, we assume that the true fraction is p and that 5% or fewer of samples would have produced a result as deviant as that observed. When binominal sampling is assumed, p(0.05) = 0.0065. Second, we make the less conservative assumption that 50% of samples would produce a result as deviant as that observed. Then p(0.50) = 0.0015. Of course, the best estimate based upon the data at hand is p = 0.0, i . e . , absolute conservation of the surveyed nucleotides among the lineages compared.

Vuriatioti among sequences homologous to PMCSl: All 12 samples from the USDA world collection of pearl millet gave identical fragment patterns for sequences homologous to pMCS1 when digested with the seven six-base enzymes listed in Table 2. Comparisons between species revealed different distributions of fragment patterns for the enzymes EcoRI, HpnI and BamHI, whereas the remaining four enzymes gave identical fragment distributions. Table 3 tabulates the estimated fragment distributions for all digests observed to vary.

Consider first the digest with BamHI. All species of Pennisetum have an identical fragment distribution that differs from Cenchrus in having a 14.1- kbp fragment and lacking a 10.0- and a 5.0-kbp fragment. The error associated with estimating the size of large fragments on agarose gels is approximately 10%; hence, the 14.1-kbp fragment may differ from the 10.0- and 5.0-kbp fragments by a single BainHI site. Reference to the BainHI map (Figure 2) shows that the 2.9-kbp band represents the comigration of three fragments: a duplicate pair of fragments from the reverse repeat and a fragment from the small single-copy region.

The EcoRI digests produce three different DNA fragment distributions. P. aineriranuin has a 1.5-kbp fragment absent in the other distributions, which have instead 0.9- and 0.5-kbp fragments. We infer that a single EcoRI site separates these fragments. P. americnnuin, P. purpureum and C. setigvrus all have

CHLOROPLAST DNA VARIATION 455 TABLE 3

Chloroplast DNA fraginrnts obserued using pMCS1

EcoRI HpaI BamHI

Frag- Frag- Frag- ment a f,o,s p,Cs ment a,f,o p,s Cs ment a,f,o,s,p Cs

A 12.0 12.0 12.0 A 10.0 10.0 10.0 A 14.1 B 2.6 2.6 B 7.6 7.6 7.6 B 10.0 C 2.4 c 5.3 C 6.8 6.8 D 2.1 2.1 2.1 D 2.7 2.7 D 5.0 E 1.5 E 2.6 2.6 2.6 E 2.9 2.9 F 1.2 1.2 1.2 F 2.0 2.0 F 1.5 1.5 G 0.9 0.9 H 0.6 0.6 0.6 I 0.5 0.5 J 0.4 0.4 0.4 K 0.3 0.3 0.3

DNA fragments are in kilobase pairs. Letters indicate the species with the tabulated fragment distribution. See Table 1 for species identification by letter.

a 2.6-kbp fragment but lack the 2.4-kbp fragment present in the other species. We have been unable to detect the putative 0.2-kbp fragment and cannot exclude the possibility that this difference results from an addition or deletion of a DNA sequence.

Finally, consider the HpaI digest. There are two patterns within the Penni- setum species. The first pattern shows a 5.3-kbp fragment absent in P. pur- pureuin and P. setaceuin, which have fragments of 2.7 and 2.0 kbp instead. The 0.6-kbp difference between these digests is well beyond the range of measure- ment error and may reflect multiple site differences or an addition or deletion event.

All of the variant sites observed using the 21-kbp Sal1 DNA fragment contained in pMCS1 map into the 12-kbp single-copy region. The DNA sequences located on this DNA fragment that map within the reverse repeat regions are invariant as expected from the studies using Ch9. M(cp. rDNA).

Variation f o r sequences hoinologus to Ch9 e(pECPR1): Like the previous probes, no variation in fragment distribution was observed among the 12 lines of pearl millet for sequences homologous to the 6-kbp EcoRI DNA fragment in the recombinant DNA probe Ch9. M(pECPR1). However, variation among species was observed for digests with EroRI, BainHI and HpaII. Table 4 tabulates fragment sizes of all observed patterns. Figure 3 shows the fragment distributions observed for EcoRI and HpaIl.

The analysis of DNA fragment pattern differences requires additional com- ment. For instance, the Pennisetum species exhibit a 6.0-kbp EcoRI fragment that differs from the 6.9-kbp fragment observed in C. setigerus. Because the probe containsjust 6 kbp of millet cpDNA, we infer that loss of an EcoRI site in C. setigerus bounding the clone from P. ainericanuin differentiates the two patterns. Likewise single-site differences can account for the BamHI and HpaIl patterns, except for the 0.29-kbp fragment observed in C. setigerus.


TABLE 4

Chloroplnst DIVA fragments observed using Ch9. M(pECPR1)

EcuRI HpaII EamHI

Frag- Frag- Frag- cs ment a,f,o,p,s Cs ment a,f,o,s p c s ment a,f,o,p,s

A 6.9 A 1.6 1.6 1.6 A 3.2 3.2 B 6.0 B 0.8 0.8 0.8 B 2.5 2.5

C 0.7 0.7 0.7 C 1.8 D 0.6 D 1.6 E 0.59 0.59 E 1.1 1.1 F 0.3 0.3 0.3 G 0.29 H 0.2 0.2 0.2

DNA fragments are in kilobase pairs. Letters indicate the species with the tabulated fragment distribution. See Table 1 for species identification by letter.

If we assume that all fragment pattern changes are due to nucleotide substitution, then the number of base substitutions per base pair @) can be estimated by the “fragment method” (UPHOLT 1977; NEI and Lr 1979). These estimates, which were obtained from the fraction of shared fragments over all digests (F), are reported in Table 5. Duplicate fragments, predicted from the restriction map of the Sol1 fragment in pMCS1, are counted twice in calculat- ing F. Table 5 shows that, even at the interspecific level, the fraction of base changes is small. For example, the best estimate is zero for the comparison between P. flucciduin and P. orientale. The mean estimates of p over all pairwise comparisons for each probe are p = 0.0032 k 0.001 for the SalI fragment in pMCS1 and p = 0.0075 +- 0.005 for the 6-kbp EcoRI fragment in Ch9. M(pECPR1). The difference between means is further reduced if the estimates are corrected for the fact that approximately half of the millet cpDNA sequences in the SalI fragment in pMCS1 fall within the invariant reverse repeat region. Thus, the estimates of p are less than 1% even at the intergeneric level. These estimates should be interpreted with caution, however, since we can not ascribe all fragment pattern changes to single-nucleotide substitutions. Indeed, addition/deletion events are common in comparative sequence studies of noncoding portions of the chloroplast genome (ZURAWSKI, CLEGC and BROWN 1984; TAKAIWA and SUGIURA 1982) and in comparative restricting mapping of chloroplast genomes (PALMER, SINGH and PILLAY 1983; BOWMAN, BONNARD and DYER 1983; GORDON et al. 1982).

Vurintioli for isozyme loci: The 12 lines sampled from the USDA World Col- lection of pearl millet were surveyed for variation using ten enzyme systems (IDH, GPDG, PGI, LAP, GOT, GDH, PGM, MDH, ADH and EST). Formal genetic analyses have been conducted for ADH (BANUETT-BOURRILLON and HAGUE 1979) and 6PGD (M. T. CLEGG, unpublished data). Tentative assignments of banding phenotypes to genetic loci were made for the remaining systems based on patterns of segregation in population samples. With the use of these assignments, 15 genetic loci determine the banding phenotypes ob-


3 4 5 Kbp -- I- - KbP 1 2

0.3. 0.29

EcoR I Hpsl I FIGURE 3.-Restriction endonuclease fragment patterns for sequences homologous to Ch9.

M(pECPR1). EmRl digests were separated on 0.8% agarose gels. Hpnll digests were separated on 2% agarose gels. Fragments were transferred to nitrocellulose filter paper ( I 7) and were hybridized to nick-translated probe. Samples run in each lane were: ( I ) P. nwrirnnum, (2) C. srtigtrus, (3) P. nntrricovutn, (4) P. purpurnrm and (5) C. .w/igrrus.

served for all enzyme systems. Seven loci are polymorphic in one or more of the 12 lines, yielding a crude estimate of 47% of loci polymorphic. This estimate is comparable to estimates for other cultivated and wild grass species (HAMRICK, LINHARDT and MIITON 1979). The failure to detect intraspecific variation for cpDNA sequences in pearl millet is clearly not indicative of low levels of genetic variability for nuclear sequences translated into enzymatic proteins.

DISCUSSION

The major results of this investigation are (1) restriction endonuclease sites within the reverse repeat region are highly conserved, and (2) changes in restriction fragment patterns do occur for singletopy regions. The factors

458 M. T. CLEGG, J. R. Y . RAWSON AND K . THOMAS

TABLE 5

Matrix of the per nuclrotide number of base substitutions estimated using the SalI Jragmwt in piMCSl (in boxhectd) nnd EcoRZ fragment in Ch9.M(pECPRl) (in stub)

Species Spe- cies a f 0 S P cs a

f 0.0

0 0.0

S 0.0

P 0.0049 (0.01 16)

c s 0.0070 (0.0065)

0.0030 (0.0035)

0.0

0.0

0.0049 (0.0116) 0.0070

(0.0065)

0.0030 (0.0035) 0.0

0.0

0.0049 (0.01 16) 0.0070

(0.0065)

0.0064 (0.0051) 0.0017

(0.0026) 0.0017

(0.0026)

0.0049 (0.0116) 0.0070

(0.0065)

0.0036 (0.0038) 0.0038

(0.0039) 0.0038

(0.0039) 0.0037

(0.0039)

0.0116 (0.0114)

0.0036 (0.0038) 0.0038

(0.0039) 0.0038

(0.0039) 0.0037

(0.0039) 0.0022

(0.0030)

The rows and columns are labeled with the first letter of the species name except for the last row and column, which is denoted by the first letters of the genus and species names (see Table 1). Standard errors are given in parentheses.

responsible for the conservation of the DNA sequences within the reverse repeat region are not completely understood. An important contributing factor to this conservation arises from the DNA sequences coding for the chloroplast ribosomal RNA (cp. rDNA), which are very highly conserved in evolution. We have shown, through the melting of DNA heteroduplexes formed between the cp.rDNA of the alga Euglena and the cp-rDNA of P. americanum, that the cp. rDNA is highly conserved between these phylogenetically distant organisms (RAWSON et al . 1981). However, cp.rDNA sequences account for less than 50% of the DNA studied within the reverse repeat region. Other studies of cpDNA evolution also show differential conservation of the inverted repeat region (KUNG, ZHU and SHEN 1982; PALMER, SINGH and PILLAY 1983). How- ever, in a recent study of three legume species, PALMER et al. (1983) showed that slow rates of evolution for the cp.rDNA sequences account for much of the differential conservation associated with the inverted repeat region.

The reverse repeat structure may act to maintain genetic homogeneity among these duplicate sequences. For example, it has been suggested that the reverse orientation of the repeat region prevents the loss or duplication of genetic material due to intragenomic recombination events (KOLODNER and TEWARI 1979). Under this hypothesis the two regions would not accumulate nucleotide substitutions independently, but, instead, new mutant sites would be transferred by recombination to both duplicate regions. There is some evidence from Chlamydomonas to support this suggestion (MEYERS et al. 1982). In addition, the large and small single-copy regions of the chloroplast genome of the common bean (Phaseolus vulgaris) have recently been shown to exist in two orientations with respect to one another (PALMER 1983), and a similar


situation appears to exist for the cyanelle genome of Cyanophora pamdoxa (BOH- NERT and LOFFELHARDT 1982). Two orientations are consistent with intramolecular recombination, which may occur frequently. Nevertheless, different lineages would be expected to evolve independently, and we would, therefore, expect to see differences at the interspecific or intergeneric level. Whatever the cause, the slow rate of genetic change observed for cpDNA sequences associated with the reverse repeat region opens up the possibility of using this region to infer genetic relationships among diverse plant taxa, perhaps up to the family level.

The single-copy regions of cpDNA monitored in this investigation do vary among species, but the rate of change appears to be much less than estimated for mammalian mtDNA evolution. Although there are no good estimates of divergence times among these species, there is a striking contrast between intraspecific estimates of p = 0.015 for old field mice (ADVISE, LANSMAN and SHADE 1979) and p = 0.03 for pocket gophers (AVISE et al. 1979) with the intergeneric estimate reported here @ = 0.006). Moreover, the slow rate of cpDNA evolution does not appear to be correlated with a low level of genetic variability for nuclear genes. Levels of isozyme variability are high, and changes in the basic chromosome number have occurred during the evolution of these species.

We thank WAYNE HANNA for supplying plant materials and for advice during the course of this project. Supported in part by grants from the National Science Foundation (DEB-81 18414) and the United States Department of Agriculture Competitive Grant Program (80-CRCR-1-0489).

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Corresponding editor: J. R. POWELL

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CHLOROPLAST DNA VARIATION IN PEARL MILLET AND RELATED SPECIES · various regions of the pearl millet chloroplast genome were used as hybridization probes. The first recombinant DNA