Plant Molecular Biology6: 171-177, 1986 © 1986 Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

Chloroplast genome organisation in sugar beet and maize Timothy Brears, Christopher L. Schardl* & David M. Lonsdale

Plant Breeding Institute, Maris Lane, Trumpington, Cambridge CB2 2LQ~ U.K. *Present address: Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546, U.S.A. Keywords: chloroplast DNA, cosmid cloning, recombination, sugar beet, maize

Summary The XhoI and SmaI restriction map of the chloroplast genome from the fertile cytoplasm of sugar beet has been constructed from overlapping cosmid clones. The genome was found to be typical of that of a dicotyledonous species, being 147.3 kb in size and having an inverted repeat. RbcL for the large subunit of ribulose-l,5-bisphosphate carboxylase, psbA for the 32 kD protein of the photosystem II reaction centre, and the 16S ribosomal RNA were located using heterologous probes. In both sugar beet and maize the inverted repeats recombine giving two isomeric forms of the genome.

mic male sterility in sugar beet (Beta vulgar&) and maize the chloroplast genomes from the fertile cytoplasms have been cloned into cosmid vectors to facilitate their analysis. Here we describe the physical map of the sugar beet chloroplast genome and demonstrate that the inverted repeat of both the sugar beet and the maize chloroplast genome can recombine.

Introduction The chloroplast genome of plants is present as a single circular DNA molecule of between 120 kb and 210 kb. In the majority of species studied the genome is characterised by having two inverted copies of a sequence located asymmetrically which split the genome into long and short unique regions. However, in some leguminous species such as pea (Pisum sativum) (12) and broad bean (Vicia faba) (11) no inverted repeat sequence is present (19). Dicotyledonous plants possessing the inverted repeat sequence arrangement consistently have larger chloroplast genomes than monocotyledonous species. The chloroplast genome of geranium (Pelargonium zonale) was found to be 210 kb (19) and is one of the largest chloroplast genomes so far reported, that of tobacco (Nicotiana tabacum) was found to be 160 kb in size (6) and those of spinach (Spinacia oleracea) and soybean (Glycine max) (7, 20) both approximate to 1~0 kb, whereas the chloroplast genomes of rice (Oryza sativa), wheat (Triticum aestivum), maize (Zea mays) and sorghum (Sorghum bicolor) (3, 4, 8, 13) all fall between 130 and 140 kb. As part of a programme to investigate cytoplas-

Materials and methods

Preparation of chloroplast DNA Chloroplast DNA (ctDNA) was isolated from fresh green tissue of sugar beet, genotype 3FA82 (fertile) and maize, genotype B 7 3 - N (fertile cytoplasm). Tissue was chilled in 4 × volume of extraction buffer (0.35 M sucrose, 0.05 M Tris-HC1 pH 8.0, 0.005 M EDTA, 0.1% BSA, 1 ml/l 2-mercaptoethanol) and homogenised in a Waring blender for 5 - 1 0 s. After filtration through miracloth a chloroplast pellet was obtained by centrifugation at 2000 x g for 15 mins. The pellet was resuspended in 0.35 M sucrose, 0.05 M Tris-HCl pH 8.0, 0.015 M MgC12 and incubated with 10 mg 171

172 DNase I/kg tissue for 1 h at 4°C. It was then washed three times in 0.6 M sucrose, 0.05 M TrisHC1 pH 8.0, 0.02 M EDTA. The final chloroplast pellet was resuspended in 0.05 M Tris-HCl pH 8.0, 0.02 M EDTA for lysis in 1% SDS at room temperature for 2 h. Lysates were then extracted several times in phenol equilibrated with 0.1 M Tris-HCl pH 8.0, and chloroform. Chloroplast DNA was recovered by ethanol precipitation and redissolved in 10 mM NaC1, 10 mM Tris-HCl pH 8.0, 1 mM EDTA.

Estab6shment of cosmid clone banks 10 tzg of ctDNA was partially digested with the restriction endonuclease Sau3A to prepare fragments of approximately 40 kb for cosmid cloning. Fragments produced by Sau3A digestion were loaded onto a step gradient of caesium chloride (01.19 to pl.49, 7 steps) and centrifuged at 40000 rpm for 2 h in a Sorvall AH650 swing-bucket rotor (15). After fractionation of gradients, an aliquot of each fraction was electrophoresed in a 1% agarose gel to locate the DNA of required size which was dialysed against 1 mM NaC1, 1 mM Tris-HC1 PH 8.0, 0.1 mM EDTA. Size fractionated ctDNA was then ligated into the BamHI sites of the cosmid vectors c2XBHC (a pAT153 derivative of c2XB (2)) and PHC79 (9). Packaging in vitro was as previously described (16). Cosmids were transfected into E. coli ED8767 met r-m SupE supF recA56. Recombinant clones were selected on the basis of resistance to ampicillin (100/zg/ml) and sensitivity to kanamycin (25/~g/ml) in the case of c2XBHC, and resistance to ampicillin and sensitivity to tetracycline (10 tzg/ml) in the case of PHC79.

Analysis of DNA from cosmM clones Minipreparations of DNA from cosmid clones were made as previously described (15). Restriction endonucleases were from Bethesda Research Ltd. and digests were carried out under the conditions specified by the manufacturer. Analysis of digests was by electrophoresis in horizontal submarine agarose gels. DNA was stained with ethidium bromide and visualised on a 300 nm transilluminator (UV Products Ltd.). Double-sided transfer of DNA from gels to

nitrocellulose filters was carried out by 'squash' blotting (16).

Recovery of DNA from agarose gels Electroelution of DNA was as previously described (16). Purification of electroeluted DNA was by phenol and chloroform extraction and ethanol precipitation.

Radioactive labelling of DNA and hybridisations DNA was labelled by nick translation or primer extension using (~-32p) dATP (Amersham International Ltd., >800Ci/mmole). Unincorporated nucleotides were separated from labelled DNA by chromatography on a 1.2 ml, 7 cm Sephadex G100 column. Nitrocellulose filters were prehybridised for 2 h at 65 °C in 3 × SSC, 10 × Denhardt's reagents, 0.05% SDS, 10/~g/ml herring sperm DNA (Sigma Chemicals Ltd.). Labelled DNA was denatured at 100 °C for l0 mins and then added directly to the prehybridisation medium. Hybridisations were continued for 16 h at 65 °C before washing twice in 3 x SSC, once in 0.3 x SSC. Autoradiography was with Kodak XAR-5 film at room temperature or at -80 °C using Cronex Lightening-Plus intensifier screens (Dupont).

Results

Construction of a physical map of the chloroplast genome from the fertile cytoplasm of sugar beet Minipreparations of DNA were made from 48 randomly selected recombinant cosmid clones for each vector. Digestion of the cosmid DNA with XhoI and SmaI (neither enzyme of which cleaves within the cosmid vector DNA) established overlaps of cosmids which covered most of the genome. In many cases restriction fragments could be ordered directly from restriction data, in others .this data could be obtained only by cross-hybridisation of cosmids. Digests of cosmids were analysed adjacent to digests of ctDNA to allow comparison of restriction fragments. In this way it was possible to reconstruct almost the entire genome with the exception of approximately 12 kb (Xho fragments H, T and G; Sma fragments L and E) - no c2XBHC

173 or pHC79 clones were found to contain any of these fragments. Those recombinant cosmids which contained restriction fragments found directly adjacent to fragments Xho-H and G (Sma-E and L) and which could be assumed to extend through this region of the genome were, without exception, rearranged and therefore of no use for mapping studies. The position of these uncloned restriction fragments was established unequivocally by crosshybridisation of the fragments, gel-purified from ctDNA digests, to adjacent cosmids and to digests

of ctDNA. Gel purified Sma-L hybridises to XhoH and to Xho-B (though only weakly to the latter due to the small overlap of 0.1 kb) and also to SmaI and Xho-M (Fig. 1). Hybridisation of Sma-E to Xho-H, Xho-T, Xho-G (Fig. 1) and to the vector containing fragment of cK6 (which has Xho-I and Sma-D as its most distal fragments) and of Xho-G to cK6 established circularity and the order of these uncloned fragments. For each enzyme a circular map of 147.3 kb (_+ 0.3 kb) was obtained. One sequence of approxi-

Fig. 1. Hybridisation of gel purified Sma-E (panel A) and Sma-L (panel B) to Xho (track 1), Xho-Sma (track 2), and Sma (track 3) digests of ctDNA. Sma-E hybridises to Xho-G, Xho-H, and Xho-T (off gel). Repeat junction fragment Sma-L hybridises to Xho-H and Xho-B (weakly) and to Sam-I and Xho-M which cover corresponding junction on the other repeat.

174

A

Table 1. Restriction mapping data: restriction site coordinates (A), restriction fragment lengths (B), and restriction fragment designations (C). Restriction data for XhoI

~

Xho

I

The chloroplast genuine of sugar beet

Fig. 2.

(A): Physical map of the sugar beet chloroplast genome showing the position of the inverted repeats and psbA, rbcL, and the 16S rRNA gene. (B): Recombination between the inverted repeats give rise to two isomeric forms of the genome.

Restriction data for Smal

A

B

C

A

B

C

0.00 1.28 11.80 12.60 15.91 31.14 32.66 35.22 36.02 37.74 38.97 40.38 73.80 84.80 97.20 98.61 102.72 108.42 109.10 114.75 129.98 133.29 134.09 147.30

1.28 10.52 0.80 3.31 15.23 1.52 2.56 0.80 1.72 1.23 1.41 33.42 11.00 12.40 1.41 4.11 5.70 0.68 5.65 15.23 3.31 0.80 13.21

P F R J B M K S L Q N A E D O I G T H B J R C

10.58 12.33 14.63 16.66 22.01 23.86 31.04 37.64 57.34 64.50 85.20 86.00 89.55 102.85 112.30 114.85 122.03 123.88 129.23 131.26 133.56 135.31 143.61 10.58

14.27 1.75 2.30 2.03 5.35 1.85 7.18 6.60 19.70 7.16 20.70 0.80 3.55 13.30 9.45 2.55 7.18 1.85 5.35 2.03 2.30 1.75 8.30 14.27

C P M N J O G I B H A Q K D E L G O J N M P F C

tion fragments produced by single and double digestion of cosmids with these enzymes. Restriction fragment lenghts and site co-ordinates are given in Table 1. Recombination between the inverted repeats in sugar beet and maize

mately 25 kb is reiterated in inverse orientation. This sequence spans Xho fragments B, J and R and Sma fragments G, O, J, N, M and P. The repeated sequences are separated by approximately 15 kb; this region is shown at the top of the circle in the illustration; the Xho-C-P site is taken as the zero coordinate (Fig. 2A). One repeat extends into and finishes in Xho-M and Sma-I at one side and in Xho-F and Sma-C at the other side, and the other repeat extends into and finishes in Xho-H and Sma-L at one side and Xho-C and Sma-F at the other side. The relative positions of XhoI and SmaI restriction sites were calculated from the sizes of restric-

In sugar beet two cosmids spanning the entirety of one inverted repeat were identified. These cosmids shared the same fragment of the long unique sequence and the Xho-M junction fragment as well as the internal Xho fragments of the repeat. However, they differed in their distal repeat junction fragment, cLll containing Xho-F while c16 contains Xho-C and in addition Xho-P (Fig. 3). Clearly this illustrates that recombination across the inverted repeat can occur in a manner similar to that described by Palmer (18) and that cLll and cI6 represent clones from the different isomeric forms of the genome (Fig. 2B). Several other cosmids

175 The physical m a p for t~amHI as published (13) placed Bam fragment 25 between Bam fragments 15 and 21 (13). Bam fragment 25 appears to have been misplaced and has been relocated between B a m l l and Bam2 (4). This location for Bam25 was confirmed from the analysis of chloroplast cosmids. Cosmids starting in Bam fragment 2 and extending through Bam fragments 16, 12, 15, 21, 3, etc. did not contain, nor did they hybridise to, Bam25. Cosmids containing Bam fragments 11, 8 and extending through the inverted repeat do not contain fragment 25 (unpublished data). Only two cosmids have been identified which contain Bam fragment 25 (cZmctND4, cZmctNAll); both grow extremely poorly, are difficult to maintain and give exceptionally low yields. As they contain Barn2 as well as Bam25 then the available data indicates that Bam25 is located between Bam fragments 2 and 11 (4).

Location of chloroplast genes on the sugar beet physical map The location of various genes on the chloroplast genome was determined by hybridisation of radioactively labelled gene probes to single and double digests of ctDNA with XhoI and SmaI (Fig. 2A). The following genes were located:

Fig. 3. Recombination between the inverted repeats of the sugar beet chloroplast genome, cL 11 (track 1), ctDNA (track 2), and ci6 (track 3) digested with XhoI. cLll and ci6 share fragments N, Q, L, S, K, M, B, J, and R. cLll has in addition fragment F and ci6 fragments C and P. These fragments represent the short single copy DNA in inverse orientation approached through the same repeat.

1. Gene for the large subunit of ribulose-l,5bisphosphate carboxylase (rbcL). A 670 bp M13mpl8 clone derived from the maize mitochondrial D N A sub-clone pLSH20 and having 95°7o homology to the 5 ' e n d of the maize chloroplast large subunit gene (14) was labelled by primer extension and used to probe sugar beet ctDNA. RbcL was found to be located on Xho-A and Sma-H. Hybridisation with nick translated pLSH20 showed a further homology to fragments within and adjoining the repeats, ie. to Xho-B (weakly), H and M and to Sma-L and I.

representing each isomer were subsequently identified. A similar situation was found in the maize cosmids. Three recombinant clones were identified which illustrated three of the four possible pairwise combinations of repeat junction fragments. These were Bam8-Baml, Bam8-Bam4 and Bam6-Bam4.

2. 16S ribosomal R N A gene The mitochondrial 3.2 kb BamH1 fragment cloned in pBR322 having 95°7o homology to the 3.2 kb BamH1 fragment of chloroplast D N A wich contains the ribosomal 16S R N A gene in maize (21) was labelled by nick translation. The 16S r R N A gene was found to be located within the inverted repeats on Xho-B and Sma-N and Sma-J.

176

3. Gene for the 32 kD protein of the photosystem H reaction centre (psbA) A 405 bp BamH1 internal fragment of psbA from wheat, cloned into the plasmid p E M B L 9 + , was nick translated. The probe hybridised to XhoH and Sma-L though not to Xho-M or Sma-I. PsbA must therefore reside on that part of Xho-H and Sma-L outside the repeat.

Discussion The chloroplast genome of sugar beet is similar to that of many other species. It has an inverted repeat of approximately 25 kb. The repeats are separated by unique DNA sequences of approximately 15 kb and 82 kb. In both sugar beet and maize the inverted repeats appear to be recombinationally active in that recombinant clones of the predicted isomeric forms of the genome have been identified. The frequency of recombination between the inverted repeats was not assessed directly but, as similar numbers of clones were identified from each isomer, it can be assumed that the isomeric forms occur in approximately equal stochiometry as in other cloroplast genomes where this genomic interconversion has been shown to occur (1, 18). The significance and mechanism of this high frequency isomerization is unclear, although in species such as pea and bean the loss of one of the two inverted repeats is correlated with both the rate of sequence drift and the rate of genomic rearrangement (17, 19). The inverted repeat may therefore play a role in the maintenance and stability of the genome. In sugar beet the inverted repeat carries the 16S ribosomal RNA gene and it is likely that the rest of the ribosomal RNA operon is also located on the repeat as in maize (5) and many other species (6, 20). The long single region of the genome carries rbcL and psbA in approximately the same positions as they are found in wheat. In maize and sorghum psbA is situated at a greater distance from the inverted repeat. In maize (Lonsdale, unpublished data) and wheat (Bowman, personal communication) the 3' non-coding region adjacent to rbcL hybridises to the junctions of the inverted repeat and the long single copy DNA. This homology was also evident in sugar beet as the maize mitochondrial rbcL

clone (pLSH20) hybridised to the repeat junctions, but an rbcL coding region probe did not. This cross hybridisation also illustrates the conservation of these minor repeated sequences between monocot and dicot chloroplast genomes. The role of these sequences is not known though it has been speculated that they are involved in the sequence inversions that characterise the chloroplast genomes from different plants. Recently, it has been demonstrated that one such minor repeat in wheat has a sequence which resembles and functions as an ATT lambda phage integration - recombination sequence (10). Despite the analysis of 96 cosmid clones using two different cosmid vectors covering some 4000 kb in total, it was not possible to clone one particular area of the sugar beet genome (Xho-H, T, G; Sma-L, E). This area includes psbA adjacent to the repeat and a further 10 kb (approximately) distal to the repeat. Difficulty in the cloning of this particular region (encompassing BamHI fragments 2 and 25) was also encountered in maize where only two cosmids out of 96 analysed spanned this region of the genome. Similarly, in wheat the 5' end of psbA (ie. that part distal to the repeat) was found difficult to clone into an M13 vector (M.D.C. Barros, personal communication). This region of the genome may contain sequences which reduce the fitness of E. coli.

Acknowledgements We would like to thank M.D.C. Barros for her gift of the psbA clone from wheat. T.B. acknowledges the support of an AFRC research studentship. C.L.S. was supported by a DeKalbPfizer Fellowship. We are grateful to Pioneer HiBred International for supplying the maize B 7 3 - N seed.

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13. Larrinua IM, Muskavitch KMT, Gubbins-EJ, Bogorad L: A detailed restriction endonuclease site map of the Zea mays plastid genome. Plant Mol Biol 2:129- 140, 1983. 14. Lonsdale DM, Hodge TP, Howe CJ, Stern DB: Maize mitochondrial DNA contains a sequence homologous to the rihulose-l,5-bisphosphate carboxylase large subunit gene of chloroplast DNA. Cell 34:1007- 1014, 1983. 15. Lonsdale DM, Hodge TP, Stoehr PJ: Analysis of the genome structure of plant mitochondria. Methods in Enzymol 118: (In press), 1986. 16. Maniatis T, Fritsh EF, Sambrook J: Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, 1982. 17. Palmer JD, Thompson WF: Chloroplast DNA rearrangements are more frequent when a large inverted repeat sequence is lost. Cell 29:537-550, 1982. 18. Palmer JD: Chloroplast DNA exists in two orientations. Nature 301:92-93, 1983. 19. Palmer JD: Evolution of chloroplast and mitochondrial DNA in plants and algae. In: MacIntyre RJ (ed) Monographs in Evolutionary Biology: Molecular Evolutionary Genetics. Plenum Publishing Corporation, 1984. 20. Spielman AA, Ortiz W, Stutz E: The soybean chloroplast genome: construction of a circular restriction site map and location of DNA regions encoding the genes for rRNAs, the large suhunit of rihulose-l,5-bisphosphate carboxylase and the 32 kD protein of the photosystem II reaction centre. Mol Gen Genet 190:5- 12, 1983. 21. Stern DB, Lonsdale DM: Mitochondrial and chloroplast genomes of maize have a 12 kh sequence in common. Nature 299:698 702, 1982.

Received 24 September 1985; in revised form 12 November 1985; accepted 19 November 1985.