Cucurbit Genetics Cooperative Report 7:66-68 (article 29) 1984

Potential of Restriction Endonuclease Analysis of Chloroplast DNA for the Determination of Phytogenetic Relationships among members of Cucurbitaceae

Juvik, J.A.

Department of Horticulture, University of Illinois, Urbana, IL 61801

J.D. Palmer

Department of Zoology, Duke University, Durham, NC 27706

A preliminary study was undertaken to determine the feasibility of using comparative restriction endonuclease analysis of chloroplast DNA as a method of accessing evolutionary relationships among members of the Cucurbitaceae. The size of the chloroplast genome of vascular plants [120-180 kilobase pairs (kb) (2)] is small enough to permit resolution of all the fragments produced after digestion of chloroplast DNA by many 6 base endonucleases and sufficiently large for rapid screening of a great many restriction sites using only a few enzymes (4). Previous studies (1,3) have indicated that changes in restriction patterns of the chloroplast genome are generally the result of base substitutions rather than major sequence rearrangements. These properties and the evolutionary conservatism of the chloroplast genome make it an excellent tool to study phylogenetic relationships among plant species, genera, and families (1,4,5).

Chloroplast DNA of 12 accessions from 11 species within 4 genera of Cucurbitaceae (Table 1) were purified according to the sucrose gradient method described by Palmer and Thompson (2). This method yielded relatively pure chloroplast DNA which was nearly free of both mitochondrial antinuclear DNA contamination. The chloroplast DNAs were digested with 9 different restriction endonucleases procured from Bethesda Research Laboratories and New England Biolabs using the instructions provided by the suppliers. The enzymes used were Sal I, Pvu II, Pat I, Sac I, Sac II, Kpn 1, Hind III, Ham HI, and Eco RI. Fragments were then separated on 0.7% - 1.0% horizontal agarose slab gels of 0.4 x 20 x 22 - 40 cm in 100 mM Tris-HCl, pH 8.1/12.5 mM NaOAc/0.25 mM EDTA.

Table 1. Numbers and size range of restriction endonuclease fragments fran the chloroplast DNA of 12 different accessions of Cucurbitaceae.
Genus/species Cultivar	Sal I		Pvu II		Sac II		Pst I		Kpn I		Sac I		Hind III		Bam HI		Eco RI
	No.	Size range (kb)	No.	Size range (kb)	No.	Size range (kb)	No.	Size range (kb)	No.	Size range (kb)	No.	Size range (kb)	No.	Size range (kb)	No.	Size range (kb)	No.	Size range (kb)
Cucurbita pepo Fordhook zucchini	5	2.2-37	8	6.8-37	9	1.7-29	11	1.0-30	13	0.8-36	21	0.7-25	25	0.4-14	36	0.4-17	39	0.6-14
Cucurbita pepo Accession from Africa	5	2.2-37	8	6.8-37	9	1.7-29	11	1.0-30	12	0.8-36	19	0.7-25	24	0.4-14	35	0.4-17	37	0.6-14
Cucurbita maxima Hubbard squash	5	2.2-37	8	6.8-37	9	1.7-29	11	1.0-30	12	0.8-36	19	0.7-25	23	0.6-14	36	0.4-17	39	0.6-14
Cucurbita mixta Gold stripe cushaw	5	2.2-37	8	6.8-37	9	1.7-29	11	1.0-30	13	0.8-36	20	0.7-25	23	0.4-14	35	0.4-17	36	0.6-14
Cucurbita moschata Butternut squash	5	2.2-37	8	6.8-37	9	1.7-29	11	1.0-30	13	0.8-36	20	0.7-25	23	0.4-14	35	0.4-17	37	0.6-15
Cucurbita andreana Accession from Mexico	5	2.2-37	8	6.8-37	9	1.7-29	11	1.0-30	11	0.8-36	19	0.7-25	24	0.6-14	34	0.4-17	49	0.6-15
Cucurbita ficifolia Accession from Mexico	6	2.2-37	8	6.8-37	9	1.7-29	10	1.0-30	15	0.8-36	19	0.7-25	23	0.4-14	34	0.4-17	37	0.6-14
Cucurbita sororia wild accession	5	2.2-37	8	6.8-37	9	1.7-29	11	1.0-30	13	0.8-36	20	0.7-25	23	0.4-14	33	0.4-17	-	-
Cucumis sativus Beit Alpha Mt.	7	2.2-21	8	2.3-37	9	1.7-29	-	-	13	0.8-36	15	0.7-25	23	0.6-13	34	0.4-17	32	0.6-15
Cucumis melo Harvest queen muskmelon	10	2.1-37	10	1.9-37	8	1.7-29	15	1.6-37	17	0.8-36	15	0.9-33	22	0.4-14	34	0.4-17	35	0.6-15
Citrullus lanatus Charleston grey	8	2.2-37	10	1.9-37	9	1.7-31	12	1.0-33	12	0.8-36	14	0.9-25	19	0.5-14	35	0.4-17	32	0.6-15
Lagenaria siceraria Bottle gourd	8	2.2-37	10	1.9-37	8	4.3-33	12	1.0-37	16	0.8-36	16	0.9-25	19	0.6-14	38	0.4-17	34	0.6-15

Table 1 lists the plant accessions used in this study and the numbers and size ranges of fragments generated by restriction endonuclease digestion. Fragment sizes for each of the digests were estimated by comparison with a control consisting of a mixture of fragments of known kilobases. Summing fragment sizes for each digest provided an estimate of the size of the chloroplast genome of each accession. Estimates ranged from 150- 160 kb for all the accessions indicating an absence of significant variation in chloroplast DNA size. Variation in the number and individual fragment size was found to exist among the different chloroplast DNAs for each of the 9 restriction enzymes. Variation in fragment size and number is indicative of mutational events at restriction sites. For accessions within the genus Cucurbita no mutations were detected at the restriction sites specific to Pvu II and Sac II. For Sal I and Pst I only the C. ficifolia accession displayed mutations at cleavage sites specific to these endonucleases. Only C. pepo, C. mixta and C. moschata were identical in digestions using Kpn I. With Sac I, C. maxima chloroplast DNA was found to be homologous to C. andreana while C. mixta, C. moschata, and C. sororia also displayed identical cleavage patterns. All the Cucurbita species had distinct fragmentation patterns when digestions were conducted using Hind III, Ham HI, and Eco RI. A substantial number of restriction site mutations were found to exist between members of the four genera tested. Only the accessions Citrullus lanatus and Lagenaria siceraria had identical cleavage sites and fragment sizes when digested by the endonuclease Sal I.

The results of this study indicate: 1) that the method employed for chloroplast DNA purification is effective for members of the Cucurbitaceae; 2) that there appears to exist both substantial variation and sufficient homology within the family to map the chloroplast genome; and 3) it should be possible through the use of parsimony analysis of shared mutations to construct a maternal phylogeny for plant species within the Cucurbitaceae.

Literature Cited

1. Kamppa, G.K. and A.J. Bendich. 1979. Chloroplast DNA sequence homologies among vascular plants. Plant Physiol. 63:660-668.
2. Palmer, J.D. 1982. Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa. Nucl. Acids Res. 10:1593-1605.
3. Palmer, J.D. and W.F. Thompson. 1982. Chloroplast DNA rearrangements are more frequent when a large inverted repeat sequence is lost. Cell 29:537-550.
4. Palmer, J.D. and D. Zamir. 1982. Chloroplast DNA evolution and phylogenetic relationships in Lycopersicon. Proc. Natl. Acad. Sci. 79:5006-5010.
5. Vedel, F., Lebacq, P. and F. Quetier. 1980. Cytoplasmic DNA variation and relationships in cereal genomes. Theor. Appl. Genet. 58:219-224.