cpDNA Probes Research Articles

Chloroplast DNA inheritance in Populus

The inheritance of chloroplast (cp) DNA was examined in F1 hybrid progenies of two Populus deltoides intraspecific controlled crosses and three P. deltoides × P. nigra and two P. deltoides × P. maximowiczii interspecific controlled crosses by restriction fragment analysis. Southern blots of restriction digests of parental and progeny DNAs were hybridized to cloned cpDNA fragments of Petunia hybrida. Sixteen enzymes and five heterologous cpDNA probes were used to screen restriction fragment polymorphisms among the parents. The mode of cpDNA inheritance was demonstrated in progenies of P. deltoides × P. nigra crosses with 26 restriction fragment polymorphisms of cpDNA differentiating P. deltoides from P. nigra, as revealed by 12 enzyme-probe combinations, and in progenies of P. deltoides × P. maximowiczii crosses with 12 restriction fragment polymorphisms separating P. deltoides from P. maximowiczii, as revealed by 7 restriction enzyme-probe combinations. In all cases, F1 offspring of P. deltoides × P. nigra and P. deltoides × P. maximowiczii crosses had cpDNA restriction fragments of only their maternal P. deltoides parent. The results clearly demonstrated uniparental-maternal inheritance of the chloroplast genome in interspecific hybrids of P. deltoides with P. nigra and P. maximowiczii. Intraspecific P. deltoides hybrids also had the same cpDNA restriction fragments as their maternal parent. Maternal inheritance of the chloroplast genome in Populus is in agreement with what has been observed for most other angiosperms.

Comparison of Restriction Fragment Length Polymorphisms in Chloroplast DNA of Five Leafy Spurge (Euphorbiaspp.) Accessions

Chloroplast DNA (cpDNA) restriction fragment length polymorphisms (RFLPs) were analyzed to assess genetic variation and relatedness among selections of North American and Eurasian leafy spurge. Leafy spurge accessions from Nebraska, Montana, Russia, Italy, and Austria were evaluated. Total DNA was extracted from young leaves and digested with the restriction endonuclease,EcoRI. CpDNA fragment patterns were determined by Southern blot analysis using mung bean cpDNA probes. Colinearity between the mung bean and leafy spurge chloroplast genomes was indicated by the observation that common fragments were hybridized by adjacent probes. Minimum estimates of chloroplast genome size for the five leafy spurge accessions, which ranged in kilobase size from 130 to 132, were within the size range of most terrestrial plants. Structural collinearity and reasonable estimates of chloroplast genome size provided evidence that the mung bean cpDNA library was suitable for characterizing leafy spurge cpDNA. Seven of the 13 mung bean probes hybridized to polymorphic leafy spurge cpDNA fragments. Based on number of polymorphisms unique to each Eurasian accession, the Austrian accession appeared to be most divergent followed by the Italian and Russian. The North American accessions seem to be most closely related to each other and to the Russian leafy spurge accession.

Molecular systematics of Brassica and allied genera (Subtribe Brassicinae, Brassiceae) —chloroplast genome and cytodeme congruence

Chloroplast DNA restriction sites for 20 endonucleases were mapped using cpDNA probes from Brassica juncea and site variation was surveyed in 33 diploid taxa of the Subtribe Brassicinae. A total of 419 mutations was observed, including both site (i.e., gain/ loss) and fragment length (i.e., insertions or deletions); 221 (53%) mutations showed variation at the interspecific level. Phylogenetic analysis indicated a clear division of the subtribe into two ancient evolutionary lineages. These were (I) the "Nigra" lineage: Brassica nigra, B. fruticulosa, B. tournefortii, Sinapis pubescens, S. alba, S. flexuosa, S. arvensis, Coincya cheiranthos, Erucastrum canariense, and Hirschfeldia incana, and (II) the "Rapa/ Oleracea" lineage: Brassica rapa, B. oleracea ssp. oleracea and ssp. alboglabra, B. rupestris-villosa complex (B. rupestris, B. drepanensis, B. macrocarpa, B. villosa), B. barrelieri, B. deflexa, B. oxyrrhina, B. gravinae, Diplotaxis erucoides, D. tenuifolia, Eruca sativa, Raphanus raphanistrum, R. sativus, and Sinapis aucheri. In the "Nigra" lineage, Brassica nigra was most closely related to the annual Sinapis species, S. arvensis and S. alba. In the "Rapa/Oleracea" lineage, the Brassica rapa and B. oleracea genomes formed a distinct group whose closest relatives were the wild species of the B. oleracea (n=9) complex (i.e., B. rupestris-villosa complex). Species with n=7 chromosomes exist in both lineages. Hirschfeldia incana (n=7), in the "Nigra" lineage, was most closely related to Sinapis pubescens. In the "Rapa/Oleracea" lineage three taxa with n=7 - B. deflexa, D. erucoides, and S. aucheri - were closely related, advanced in the lineage, and were the closest apparent relatives (particularly D. erucoides) to B. rapa, B. oleracea, and its wild relatives. Levels of genetic divergence suggested by the cpDNA data were consistent with cytodeme recognition in the subtribe, but provided evidence for inconsistencies in the current generic delimitations based on morphology. Very low levels of genetic divergence were evident among taxa/accessions within a cytodeme. Raphanus was closely related to the Brassica rapa and B. oleracea genomes and clearly belongs in Subtribe Brassicinae. Several cytoplasmic genetic markers of potential use in plant breeding programs were identified for each of the cytodemes.

Inheritance of chloroplast DNA inPopulus

Restriction fragment length polymorphisms (RFLPs) were used as markers to determine the transmission of chloroplast DNA (cpDNA) in poplar crosses. The plant material studied included individual trees ofPopulus trichocarpa, P. maximowiczii xtrichocarpa, P. maximowiczii xnigra, and offspring from controlled crosses between these trees. RFLPs were identified by direct observation of stained restriction fragments, as well as by molecular hybridization with heterologous cpDNA probes. Analysis of the restriction fragment patterns in the parents and their progeny showed only the patterns of the maternal tree in the progeny, while no paternal type was found. These results provide clear evidence of a maternal mode of chloroplast inheritance in the poplar clones studied.

Restriction Site Variation in the Chloroplast Genome of Sorghum (Poaceae)

Restriction site variation was analyzed in the chloroplast (cp) DNA's of six species of Sorghum and one species each of Cleistachne and Zea. Each cpDNA was separately digested with 14 restriction enzymes and hybridized with six maize or sorghum cpDNA probes that together comprise approximately 75% of the plastid genome. Eighty-four restriction site mutations and two deletion mutations were observed among the cpDNA's of these species. These variable character mutations were used in phylogenetic analyses to construct rooted, most-parsimonious, phylogenetic trees on which confidence limits were placed by bootstrap analysis. The estimated number of nucleotide substitutions per site between each of the cpDNA's was also calculated. These analyses 1) reveal a relatively high level of sequence divergence within the genus Sorghum relative to that of other genera; 2) confirm that Sorghum sect. Sorghum is a monophyletic group; 3) cast doubt on the monophyly of sect. Parasorghum; and 4) suggest that the genus Sorghum may be either para- phyletic or polyphyletic. The fact that restriction site presence/absence in the plastid genome could be easily studied between genera as divergent as Zea and Sorghum suggests that analysis of variation in cpDNA could be used to construct a phylogeny for the entire tribe Andropogoneae.

Adiantum capillus-veneris Chloroplast DNA Clone Bank: As Useful Heterologous Probes in the Systematics of the Leptosporangiate Ferns

Restriction fragment length polymorphism (RFLP) analysis of chloroplast DNA (cpDNA) has been applied successfully to solve systematic problems in various groups of angiosperms (reviewed in Palmer, 1987, 1988). It is now widely accepted that this method can provide more definite evidence of systematic relationships than can morphological, cytological, or chemical (secondary plant products) analyses. Two major approaches are used to detect cpDNA RFLPs. One is that of preparing purified cpDNA (Palmer & Zamir, 1982; Ogihara & Tsunewaki, 1982; Palmer et al., 1983), and the other is that of preparing total cellular DNA, in which cpDNA RFLPs are detected by means of heterologous cpDNA probes (Sytsma & Schaal, 1985; Sytsma & Gottlieb, 1986a,b; Jansen & Palmer, 1988). Since, in pteridophyte molecular systematics, we usually use field-collected living materials and often have to extract DNA from small quantities of leaf tissue, the second method is the more suitable. In contrast to the wide use of cpDNA analysis for angiosperm systematics, for pteridophyte cpDNA systematic studies only two groups, Osmunda (Stein et al., 1986) and some polystichoid ferns (Yatskievych et al., 1988; Stein et al., 1989; Soltis et al., 1990), have been analyzed. These pioneering studies have clearly demonstrated the usefulness of cpDNA analysis in pteridophyte systematics. One problem encountered when applying RFLP analysis of cpDNA to pteridophyte systematics is that currently available angiosperm heterologous cpDNA probes hybridize to fern cpDNA only weakly, even in low-stringency conditions, and give only slight signals. Improvement of probe DNA is thus desirable to ensure stronger signals. This will be possible by preparing better-matched probes from fern cpDNAs, since the maximum rate of hybridization at the optimal temperature is expected to increase as the homology between probe and sample DNAs increases (Bonner et al., 1973; Anderson & Young, 1985). In this paper, we report application of our newly prepared Adiantum capillus-veneris cpDNA clone bank (Hasebe & Iwatsuki, 1990) to pteridophyte systematics. This is the first nearly complete clone bank from a pteridophyte that has been characterized by both physical and gene maps.

Chromosomal localization of ceruloplasmin and transferrin genes in laboratory rats, mice and in man by hybridization with specific DNA probes.

Fragments of the natural rat ceruloplasmin (Cp) gene and cDNA copies of rat Cp and transferring (Tf) mRNAs highly labelled by nick translation with 125I-dCTP were used as specific probes for assignment of these genes to the metaphase chromosomes of rat, mouse and man by in situ hybridization. Both Cp and Tf genes were found to be syntenic in rodents, occupying with high probability the regions 9D and 9F1-3 in mice and 7q11-13 and 7q31-34 in rats respectively. The significant increase in silver grain count over chromosome 15 in rats after hybridization with both the Cp and Tf probes suggests the presence of a related pseudogene cluster on this particular chromosome and thus favours its partial homeology to chromosome 7. The localization of silver grains in metaphase chromosome of man indicates subregional assignment of the Tf gene to 3q21. Use of the rat Cp DNA probe does not indicate synteny of the Cp and Tf genes in man and suggests the existence of a related DNA sequence in 15q11-13. The potential and limitations of the in situ hybridization technique with heterologous DNA probes for gene mapping in mammalian species are discussed.

Independent segregation of the plastid genome and cytoplasmic male sterility in Petunia somatic hybrids

The chloroplast genomes of three sets of Petunia somatic hybrids were analyzed to examine the relationship between chloroplast DNA (cpDNA) composition and cytoplasmic male sterility (CMS). Chloroplast genomes of somatic hybrid plants were identified either by restriction and electrophoresis of purified cpDNAs or by hybridization of total DNA digests with cloned cpDNA probes that distinguish the parental genomes.