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Variation in the psbC gene region of gymnosperms and angiosperms as detected by a single restriction site polymorphism
Abstract
This paper reports on a PCR-RFLP analysis in a chloroplast DNA region consisting of coding and intergenic spacer sequences
of trnS and the adjacent psbC gene. This region was PCR-amplified in 62 woody plant species, predominantly tree species, that represent a broad systematic
range in both gymnosperms and dicotyledonous angiosperms. The amplification products were digested by the restriction endonuclease
HaeIII (GG↓CC). Fourteen different restriction patterns occurred, 5 of which characterised representatives of the gymnosperms,
and 9 angiosperm representatives. A single restriction site polymorphism revealed most of the species to share restriction
patterns. Groups formed which showed relationships to plant systematic units. This phenomenon is discussed with regard to
the psbC gene and the GGCC motif for tracing species’ relationships on a high taxonomic level of gymnosperms and angiosperms.
... Earlier ex-periments on Abies alba by PCR-amplification of a cpDNA region consisting of both the gene and intergenic sequences of the trnS and psbC (trnS-psbC) followed by restriction digestion (PCR-RFLP) with 14 enzymes regions, revealed that this region had no sites for any of the enzymes tested except HaeIII (Ziegenhagen et al. 1995). Subsequently, the screening of 62 woody plant species (34 species of gymnosperms and 28 species of angiosperms) for single-restriction site (GG↓CC) polymorphisms (Ziegenhagen and Fladung 1997), as well as some non-woody annuals (unpublished), demonstrated the utility of this system to differentiate groups at higher taxonomic levels than species. It has been presumed that amplifying the trnS-psbC cpDNA region from wideranging species, as well as the application of this technique to other chloroplast genes, would be worthwhile. ...
... The present study on PCR-RFLP of the chloroplast gene region trnS-psbC with HaeIII in 24 species of mangrove and mangrove-associate species, belonging to 11 orders, 13 families and 17 genera of angiosperms, revealed 18 classes of restriction patterns. This was more than the 13 restriction patterns observed in 34 species of gymnosperms and 28 species of angiosperms representing 14 orders (3 gymnosperms and 11 angiosperms), 20 families (7 gymnosperms and 13 angiosperms) and 41 genera (20 gymnosperms and 21 angiosperms) with the same cpDNA region and restriction enzyme reported previously by Ziegenhagen and Fladung (1997). Moreover except for one genus, Abies of the gymnospermae, in which intra-generic and intra-specific variation was reported, the 14 (G5+A9) banding patterns obtained by Ziegenhagen and Fladung (1997) were different only at the genus level. ...
... This was more than the 13 restriction patterns observed in 34 species of gymnosperms and 28 species of angiosperms representing 14 orders (3 gymnosperms and 11 angiosperms), 20 families (7 gymnosperms and 13 angiosperms) and 41 genera (20 gymnosperms and 21 angiosperms) with the same cpDNA region and restriction enzyme reported previously by Ziegenhagen and Fladung (1997). Moreover except for one genus, Abies of the gymnospermae, in which intra-generic and intra-specific variation was reported, the 14 (G5+A9) banding patterns obtained by Ziegenhagen and Fladung (1997) were different only at the genus level. These observations provided molecular evidence for the highly diverse nature of the mangrove gene pool based on morphological characters (Blasco 1984). ...
Chloroplast DNA (cpDNA) regions, trnS-psbC and rbcL, from 120 individuals of 24 mangrove and mangrove associate species belonging to 11 orders, 13 families and 17 genera of
Angiospermae were amplified by the polymerase chain reaction (PCR) and restriction-digested with HaeIII. Analysis of polymorphism in the restriction fragments (PCR-RFLP) revealed 18 classes of restriction banding pattern in
trnS-psbC region. This has provided molecular evidence for diversity in the mangrove floral component at the above-species level.
Intra-generic variations were observed in three genera, viz. Rhizophora, Avicennia and Suaeda. Species-specific restriction patterns were found in the genera Rhizophora and Suaeda. A natural hybrid belonging to the genus Rhizophora was also analysed, and its restriction pattern was the same as that of a putative parental species.PCR-RFLP analysis of rbcL gene region was less differentiating. However, it showed 13 different classes of restriction patterns and revealed the usefulness
of these investigations for genome analysis at a higher taxonomic level. Intra-specific variation was not observed in any
of the species in either of the cpDNA regions analysed. This is the first report which describes variations in the chloroplast
genome of mangrove species.
... Despite the biological and economical importance of the Abies species occurring in this region, current knowledge on their relationships is based mainly on morphological, anatomical and biochemical studies (Liu 1971;Mitsopoulos and Panetsos 1987;Farjon and Rushforth 1989;Fady et al. 1992;Fady and Conkle 1993). Little is known about the genetic variation among these Abies species at the DNA level (Vicario et al. 1995;Vendramin and Ziegenhagen 1997;Ziegenhagen and Fladung 1997). ...
... pinsapo. Furthermore, to allow more detailed analysis of the region between the trnS and the psbC genes that showed intra-speci"c polymorphism in A. alba (Ziegenhagen et al. 1995;Ziegenhagen and Fladung 1997), we increased the sample sizes of the taxa for which additional material was available (A. alba, A. nebrodensis and Abies pinsapo var. pinsapo) (see Table 1). ...
... The results from the analysis of the trnS-psbC region were concordant with those of Ziegenhagen et al. (1995) and Ziegenhagen and Fladung (1997). As expected, after digestion of the region with HaeIII, we detected two variants in A. alba evenly present among the 26 individuals analysed. ...
We used PCR-RFLP analysis of the chloroplast DNA of the genus Abies (family Pinaceae), to determine if the method could be employed to detect inter-specific variation in this genus and to study how the variation
was distributed in different regions of the genome. Ten different chloroplast DNA regions, consisting of coding and non-coding
DNA sequences, were amplified with specific primers in ten different Abies taxa. The amplification products were digested with several restriction enzymes. The results showed that the chloroplast
genome is highly variable in most of the investigated taxa and contains multiple variable regions that appear to be distributed
throughout the whole genome. Species-diagnostic markers were found for four of the ten investigated species. Unexpectedly,
intra-specific variation was also detected in four species. It is likely that further studies, including larger sample sizes
and/or more powerful methods for the detection of chloroplast DNA variation, will reveal additional variation for this genus.
... In Abies, the multiple variable regions are believed to be scattered throughout the whole genome (PARDUCCI and SZMIDT, 1999). Several attempts to characterize such variation in the genus Abies using PCR-RFLP approach have been attempted in recent years (TSUMURA et al., 1995(TSUMURA et al., , 2000ZIEGENHAGEN and FLADUNG, 1997;PARDUCCI and SZMIDT, 1999). The results showed unequivocally the variable nature of Abies cpDNA especially at the species level. ...
... The results showed unequivocally the variable nature of Abies cpDNA especially at the species level. However, within-species variation has also been documented in A. alba, A. mariesii and A. sachalinensis (ZIEGENHAGEN and FLADUNG, 1997;TSUMURA et al., 1994;HAYASHI et al., 2000). This aspect should be kept in mind when interpreting the results of presented study involving only 1 specimen of each species investigated. ...
Using PCR-RFLP analysis, a comparative study on the restriction site polymorphism within 8 genes and regions of the Abies chloroplast DNA has been conducted covering 15 Asian, 6 North American and 7 Mediterranean species. A variable degree of divergence was observed among individual species of a given region as well as between geographical groups. A group of the Mediterranean firs, consisting of closely related species, differed profoundly from both Asian and North American representatives. Although a higher level of restriction site variants was detected among the Asian firs, two thirds of them were allocated to the difference between A. mariesii and the other Asian firs. The North American species exhibited the highest level of polymorphism resulting in several subgroups on a cladogram. At the individual species level, the Asian species A. mariesii and the North American species A. lasiocarpa diverged conspicuously from their counterparts in their respective regions. The results of restriction site polymorphism analysis are discussed with ragard to crossability and taxonomic status of individual species.
... Primers developed by Demesure et al. (1995) have been proved to amplify in many plant species. For instance, Ziegenhagen and Fladung (1997) were able to PCRamplify the chloroplast intergenic spacer trnS-psbC (Demesure et al. 1995) in 62 woody plant species that represented a broad systematic range in both gymnosperms and dicotyledonous angiosperms. Moreover, Sun (2002) indicated that published universal primers by Demesure et al. (1995) for amplification of trnK (exon 1)-trnK (exon 2) successfully amplified the respective regions in Elymus species. ...
... Different studies have been conducted to measure the genetic diversity or phylogenetic analysis of Artemisia species by using different molecular markers (Mohsen and Ali, 2008;Hayat et al., 2009;Nazar and Mahmood, 2010). Besides the other molecular markers related to techniques for phylogenetic studies, cpDNA is a genome frequently used in population genetic studies (Comes and Abbott, 1999), and it has successfully been used for phylogenetic analysis (Ziegenhagen and Fladung, 1997). Polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) has the ability to discriminate between genotypes based upon the presence or absence of restriction sites within the amplified DNA. ...
Artemisia L. of the family Astereacae is a genus with enormous economical and medicinal importance. As a result, the genus Artemisia has been the subject of diversity-focused studies. In the present study, phylogenetic analysis based on restriction fragment length polymorphism of the chloroplast rps11 gene was conducted on eight species of Artemisia that represent the major morphological subgroups. After digestion of the rps11 gene that was amplified from eight species, with six different restriction enzymes, each restriction site was observed and scored on a 12% polyacrylamide gel. The data were analyzed using the Numerical Taxonomy and Multivariate Analysis System to infer the phylogenetic relationship within the genus. A mixed pattern was observed among the species belonging to various taxonomic groups of Artemisia.
... Further digestion of their amplicons with restriction enzymes (PCR-restriction fragment length polymorphism; PCR-RFLP) is therefore conducted for displaying the restriction site polymorphisms 9 . PCR-RFLP of the rDNA-ITS 10 and cpDNA 11,12 have been extensively used for genetic diversity studies in several plants because of its simplicity, reliability, and practicality. ...
The PCR-restriction fragment length polymorphism (PCR-RFLP) approach was successfully developed
to identify 25 native Dendrobium species in Thailand. PCR-RFLP of the rDNA-ITS with six restriction enzymes and
three chloroplast (cp) DNA regions with five primer-enzyme combinations produced 24 types of DNA patterns altogether.
Twenty-three out of the 25 species determined in this study were found to belong to unique classes and were successfully
differentiated. Two species, D. crumenatum and D. formosum, possessing the same DNA pattern, however, were identified
after cutting the chloroplast DNA fragment amplified by psbC-trnS primer with MboI enzyme. An effective procedure for
identifying each Dendrobium species was developed. PCR-RFLP of the rDNA-ITS with TaqI, which is the most informative
enzyme, was used for the early detection of 16 Dendrobium species. To identify the remaining Dendrobium species, PCRRFLP
analysis was performed using one more primer-enzyme combination. Our study provides a rapid, simple, and reliable
identification method for these Dendrobium species.
... There was a high level of polymorphism among the genera (59.15 %) in all the enzyme digests, indicating a high degree of separation of the genera over evolutionary time. The amplification of the two genes in the species revealed that the size of amplified fragments is the same as that reported for other species (Ziegenhagen and Fladung 1997). No size differences were observed in any of the analysed species. ...
... This is because of the observation that the number of bands seen in Rhizophora was more than that in other species analysed. The present observations are in line with that of Ziegenhagen and Fladung (1997), wherein they observed that evolution of primitive to advanced species seems to correlate with a reduction in the number of restriction fragments obtained for the amplified gene region. The trnS-psbC gene seems to be evolutionarily relevant for Rhizophora; as a number of different patterns reflect different degrees of systematic sub-divisions in it. ...
Molecular phylogeny and genetic diversity in all the nine species (Rhizophora mucronata, R. apiculata, R. stylosa, Bruguira cylindrical, B. parviflora, B. gymnorriza, Ceriops tagal and C. decandra) and a natural Rhizophora hybrid, of the mangrove Rhizophoraceae, occurring in the Indian sub-continent were analysed using random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) and RFLP of polymerase chain reaction amplified chloroplast genes (PCR-RFLP) as markers. Intra-specific variability as revealed by RAPDs were low in all the analysed species. Inter-specific RFLP analysis revealed species-specific profiles in some probe-enzyme combinations. The rDNA repeat units, as flanked by the Hind III restriction sites was found to be very conserved within each genus and three different rDNA repeat units were observed among the four genera. Generic differences in PCR-RFLP were observed only in rbcL and trnS-psbC gene regions. Species differences were observed in Rhizophora in the PCR-RFLP of trnS-psbC and trnL-UAA with Hae III and Taq I, respectively. Rhizophora mucronata was found to be the chloroplast donor for the natural inter-specific hybrid. A dendrogram based on the data sets from all the three marker systems revealed that the four genera segregated into three species groups.
... Primers developed by Demesure et al. (1995) have been proved to amplify in many plant species. For instance, Ziegenhagen and Fladung (1997) were able to PCRamplify the chloroplast intergenic spacer trnS-psbC (Demesure et al. 1995) in 62 woody plant species that represented a broad systematic range in both gymnosperms and dicotyledonous angiosperms. Moreover, Sun (2002) indicated that published universal primers by Demesure et al. (1995) for amplification of trnK (exon 1)-trnK (exon 2) successfully amplified the respective regions in Elymus species. ...
Universal (consensus) primers are those primers that have the ability to amplify the targeted region of DNA across a broad
range of individuals in a certain group of organisms. In plants, such universal primers have been designed to target regions
in the nuclear, mitochondrial or chloroplast genome. Among these three genomes, the chloroplast genome is the most suited
for the design of consensus primers due to the lower rate of evolution and hence conservation of gene order and sequence of
the genome among the different plant species compared to the other two genomes. Several molecular studies in plants have developed
and used chloroplast-specific universal primers. In this review, I present some examples of the nuclear DNA-specific universal
primers and discuss the features of the chloroplast DNA that make it the most suited for the design of such primers. I then
refer to all chloroplast-specific primers developed so far and provide some examples of molecular studies and applications
that made use of them.
Additional key wordschloroplast genome–consensus primers–molecular systematics
... This is because of the observation that the number of bands seen in Rhizophora was more than that in other species analysed. The present observations are in line with that of Ziegenhagen and Fladung (1997), wherein they observed that evolution of primitive to advanced species seems to correlate with a reduction in the number of restriction fragments obtained for the amplified gene region. The trnS-psbC gene seems to be evolutionarily relevant for Rhizophora; as a number of different patterns reflect different degrees of systematic sub-divisions in it. ...
Molecular phylogeny and genetic diversity in all the nine species (Rhizophora mucronata, R. apiculata, R. stylosa, Bruguira cylindrical, B. parviflora, B. gymnorriza, Ceriops tagal and C. decandra) and a natural Rhizophora hybrid, of the mangrove Rhizophoraceae, occurring in the Indian sub-continent were analysed using random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) and RFLP of polymerase chain reaction amplified chloroplast genes (PCR-RFLP) as markers. Intra-specific variability as revealed by RAPDs were low in all the analysed species. Inter-specific RFLP analysis revealed species-specific profiles in some probe-enzyme combinations. The rDNA repeat units, as flanked by the Hind III restriction sites was found to be very conserved within each genus and three different rDNA repeat units were observed among the four genera. Generic differences in PCR-RFLP were observed only in rbcL and trnS-psbC gene regions. Species differences were observed in Rhizophora in the PCR-RFLP of trnS-psbC and trnL-UAA with Hae III and Taq I, respectively. Rhizophora mucronata was found to be the chloroplast donor for the natural inter-specific hybrid. A dendrogram based on the data sets from all the three marker systems revealed that the four genera segregated into three species groups.
... Digestion of purified trnS-psbC with the eight restriction enzymes individually showed the presence of at least two restriction sites for each enzyme. This is in contrast with earlier reports in the species of Abies (Ziegenhagen et al. 1995;Ziegenhagen and Fladung 1997), and Cajanus and its allied genera where many enzymes did not have sites at all. ...
The chloroplast trnS-psbC gene regions from total genomic DNA of 119 accessions from seven small millet species were amplified by polymerase chain reaction (PCR) and digested with eight restriction enzymes individually as well as in combinations of two enzymes to generate restriction fragment length polymorphism (PCR-RFLP). PCR-RFLP with individual enzymes revealed polymorphism between only some species. However, all the species could be distinguished by using a combination of two enzymes, specifically HaeIII and MspI. PCR-RFLP of 11 to 20 accessions with the same enzyme combination showed no intraspecific variation, which established that the differential banding patterns were species specific. In contrast, the same enzyme combination was not useful for differentiating different species of the genera Cajanus, Rhyncosia, Abies, Rhizophora, Ceriops, and Bruguiera, and it also revealed intraspecies variation in three species of Abies. The present study indicated that digestion of trnS-psbC with two four-base recognizing enzymes reveals more variation than with either enzyme alone and that it may be a method of choice for species identification in some genera.
To identify intergeneric, interspecific, and intraspecific polymorphisms, PCR-RFLP studies of the chloroplast rbcL gene profiles were carried out using five restriction endonucleases (AluI, HpaII, BsuRI, BstHHI, BstMBI) in 14 species belonging to 6 different genera and 7 Thuja occidentalis cultivars. For all the genera studied (Pinus sp., Abies sp., Picea sp., Microbiota sp., Syringa sp., Thuja sp.), A genus-specific restriction profile characterized by a unique combination of restriction DNA fragments was revealed. The combination of enzymes used in the study did not reveal the interspecies and intraspecific variability of the rbcL gene. Species belonging to the same genus and all varieties of western thuja were characterized by identical PCR-RFLP profiles. It is assumed that further research, with the inclusion of a larger number of species and enzymes, will reveal polymorphism at the interspecies and intraspecific levels.
Natural forests and forest plantations currently cover 26.6% of the total land area of the earth (FAO 1997). Forests basically affect both, carbon and water cycles on our planet and provide manifold benefits by harboring species and protecting landscapes against erosion, by supplying shelter against wind, snow, radiation or noise, and by offering various recreation purposes and outdoor activities. Economic benefits arise from utilization of timber, bark and many other natural products for various industrial purposes (mainly pulp and paper production), for energy production and for supply of food.
The divergence in the sequence of chloroplast genes is considered to proceed more slowly than the divergence of nuclear genes (1). Thus, polymorphisms of chloroplast genes have commonly been used as genetic markers for studies at above the species level. Many investigations of chloroplast gene polymorphism have been made using RFLPs (restriction fragment length polymorphisms), a procedure which involves time-consuming hybridization steps (for examples, see reference (2) and for RFLP protocols, see Chapter 6). More rapid and efficient exploitation of chloroplast DNA polymorphism can, however, be achieved by means of PCR based techniques.
The internal transcribed spacer (ITS) of ribosomal DNA (rDNA) was used as a marker to identify genetic differences among 24 accessions of species and hybrids of the Oncidiinae based on PCR-amplified RFLP analysis. The ITS region of rDNA in 24 respective accessions of the Oncidiinae was amplified by a pair of complementary primers to conserved regions at the 3’ end of 17S and 5’ end of 25S of rRNA genes. The ITS was found to be about 730 bp in length, and there was no obvious variation in length among the 24 accessions studied. The PCR-amplified samples were digested with ten restriction enzymes and subjected to electrophoresis. Among the 166 bands that appeared, 159 were polymorphic. Cluster analysis was then done based on the pattern of band distribution in all samples studied. Four major groups and two individual clusters not belonging to any of the four groups were identified based on molecular data. The results of the study provide a DNA marker that can serve as a good tool to identify new hybrids of Oncidiinae flowers at the molecular level. This technique could be applied to protect the rights of plant breeders in the future.
Genetic structure of silver fir (Abies alba Mill.) populations in Slovakia was analyzed using a PCR-RFLP approach. Based on the original finding of ZIEGENHAGEN and FLADUNG (1997), the variation in the cpDNA region consisting of coding and intergenic spacer sequences of the trnS and psbC genes was tested in 285 individuals representing 8 populations of the species. The two Hae III haplotypes have accordingly been recognized. The first haplotype was characterized by 3 restriction fragments as compared with the 4 restriction fragment-pattern of the second haplotype. The proportion of the two haplotypes varied considerably in individual populations but the prevalence of the first haplotype was observes in the East Slovakian populations Zboj and Palota. The opposite figure was characteristic for the populations in the middle and northern parts of Slovakia with a prevalence of individuals exhibiting second haplotype.
Genetic structure of silver fir primeval forests was compared with that of the adjoining stands of the species using DNA and isozyme markers. At the DNA level, the proportion of cytotypes A and B generated by digestion of trnS-psbC flanking region of chloroplast DNA (cpDNA) with Hae III was shown to differ significantly between the primeval forest in StuŽica on one side and in Dobroč and Badín on the other. Significant differences of the kind were revealed between additional 8 populations of Abies alba in Slovakia. On the contrary, the differences in cytotype proportions between the primeval forests given above and the corresponding managed stands adjoining them have not reached the level of statistical significance. At the isozyme level, the primeval forest Badín contained similar number of alleles per locus as the adjoining managed stand. The reverse relationship between the primeval and managed stands was however found at the localities Dobroč and StuŽica. The observed heterozygosity was higher in primeval forest Badín and Dobroč than in the corresponding managed stands but the reverse figure was typical for the locality StuŽica. The isozymes seem to be more efficient markers than cpDNA.
Crossability relationships between the North American species Abies concolor and five Mediterranean species of the genus Abies was analyzed using artificial hybridization approach. The species was proved to be reproductively isolated from the Mediterranean firs as evidenced by the incompatible nature of its crossings with A. alba, A. nordmanniana, A. cephalonica, A. cilicica and A. pinsapo. Gametophytic incompatibility based on inhibition of pollen tube growth in nucellar tissue of an ovule was shown to operate in preventing mutual hybridization of these species.
DNA sequences of the chloroplast spacer region between the trnL and trnF genes were determined in 18 species of Abies Mill. and another five species of Pinaceae (Keteleeria davidiana, Tsuga sieboldii, Larix kaempferi, Pseudotsuga menziesii, and Picea bicolor). A tandem repeat sequence composed of a 14-bp core sequence was found in all Abies species analyzed. This tandem repeat array was specific for genus Abies, since this array was not detected in the other five species of Pinaceae. Comparison of the tandem repeat region of Abies species revealed variation in the number of repeats and in the nucleotide sequences of the units among species. The number of repeats varied from two to five, and there were eight different unit types in nucleotide sequences. Eighteen Abies species were divided into nine groups by the tandem repeat type, which was defined by both the number of repeats and the unit type. This grouping was compared with two conventional classifications based on morphological traits. There were some differences between the results of this study and the conventional classifications.
Salinity in agricultural land is a major problem world wide, placing a severe constraint on crop growth and productivity in many regions and increased salinization of arable land is expected to have devastating global effects. Though plants vary in their sensitivity to salt stress, high salinity causes water deficit and ion toxicity in many plant species. Considerable efforts have therefore been made to investigate how genes respond to salt stress in various plants by using several approaches, including proteomics. Proteomic approaches for identifying proteins that are regulated in response to salt stress are becoming common in the post-genomics era of crop research. In this chapter, a detailed description of physiological, biochemical and antioxidative genes of salt stress in mangroves is given. Recent developments on salt stress genes of mangrove origin are also discussed and their identification applying bioinformatics approach as well as their validation in lower unicellular organisms. In addition, genomic biological changes in the proteomes of mangroves under salt stress condition are discussed. This chapter will provide a viewpoint into how proteomics and genomic based research is likely to develop in this field. Accumulation of compatible solutes and induction of antioxidative enzymes are other mechanisms of salt tolerance in mangroves. Mangroves also provide a reservoir for some of the best known, and at times, novel genes and proteins, involved in tolerance to salinity stress, that are likely at work in other plants. The salt tolerance genes listed in this review most likely represent only the tip of the iceberg, and continuous efforts to isolate and identify novel useful genes and promoters from mangroves are necessary; DNA microarray technology in particular is likely to become a powerful tool for this purpose. Eventually, the largest challenge will be to combine these genes and promoters in a systematic and logical way in order to maximize plant salinity tolerance. When realized, genetic engineering of crop and industrial plant for salinity tolerance using genes isolated from mangroves will be a vitally important tool in the quest to alleviate the earth’s future problems concerning food, energy, and the environment.
Genetic analysis enables individual identifications of trees and shrubs, the development of methods to trace back plant material in the case of false labelling or investigation of damages caused by roots of trees. This individual genetic “barcodingprocedure” is performed by using the so called M13-fingerprinting method. A further technique is highlighted (microsatellites = SSRs) in order to control the varietal identity of offspring from specific individuals, crosses and breeding programs.
Four cpDNA regions were analyzed with the use of PCR-RFLP technique and nucleotide sequences
of two mtDNA regions were characterized in order to find P. sylvestris and P. mugo species specific markers useful for studies of the species hybridization. The difference in the restriction fragment patterns of trnV-rbcL region after digestion with MvaI endonuclease was detected. The analyses of the species representatives from various geographic regions revealed that the observed polymorphism is species specific. No differences have been disclosed in the analyzed trnS-trnT, trnK1-trnK2, trnC-trnD cpDNA regions. The P. sylvestris and P.mugo mtDNA sequences of orf25 and coxI regions proved to be identical.
Variations of the chloroplast DNA (cpDNA) from three of the four cultivated species of cotton (Malvaceae); Gossypium barbadense L., Gossypium hirsutum L., Gossypium arboreum L. and its synonym Gossypium nanking Meyen., were analyzed. Using specific set of primers, the whole circular cpDNAs from the four test species were amplified.
These were subsequently digested with the use of seven restriction enzymes. The amplified fragments of the whole cpDNAs of
the diploid cultivated cotton G. arboreum and its synonym G. nanking did not show any differences. However, the allotetraploid cultivated cottons G. barbadense and G. hirsutum, showed some fragment length differences directly visible after amplification and two types of restriction fragment length
polymorphism (RFLP), the first appeared as slightly lengthened bands and the other as gain or loss of a restriction site.
The results also showed that the chloroplast genomes of the allotetraploid cultivated cottons are highly similar to the diploid
cultivated cottons tested in terms of length and digestion patterns. The detected amplified length differences, RFLPs and
the restriction sites can be considered as species specific markers for the allotetraploid cultivated cottons, which could
be a useful tool for future studies of the cpDNA of the genus Gossypium L.
The CP47 and CP43 subunits of Photosystem II bind chlorophyll a and belong to a family of (bacterio)chlorophyll-binding proteins
that serve as core antenna polypeptides in both anoxygenic and oxygenic photosynthesis. Uniquely, both of these proteins possess
extended hydrophilic domains that contribute to the environment of the oxygen-evolving complex. Structural studies have shown
that these polypeptides are associated with both intrinsic components of the reaction center and with the extrinsic proteins
that enhance O2 evolution under physiological conditions. The biochemical, spectroscopic and molecular techniques that have been used to
define these interactions, as well as the functional domains within these proteins, are presented and encompass studies performed
on photosynthetic eukaryotes and cyanobacteria.
Twenty-eight species belonging to five genera of the sub tribe Cajaninae; viz., Cajanus(15 species), Rhynchosia (10 species), Dunbaria, Flemingia and Paracalyx were analysed for variations in four chloroplast generegions, rbcL, trnS-psbC, 16S and trnL-UAA. The four chloroplast gene regions were amplified with specific primers and subsequently digested with 15 restriction
enzymes. Rhynchosia did not show any inter-specific differences in the four gene regions for any of the enzymes used. In Cajanus, the inter-specific PCR-RFLP profile of the four gene regions for all the enzymes were similar except for the Pst I digests of trnS-psbC gene region. At inter-generic level, rbcL gene region did not show polymorphism with any of the enzymes used; while in trnS-psbC gene region polymorphism was observed only with Pst I and Hae III digestions. Inter-generic PCR-RFLP of 16S did not reveal any variation. The trnL-UAA gene region had restriction site only forEcoR I in which Cajanus and Rhynchosia showed similar profiles while Dunbaria showed a different profile. The trnL-UAA gene region in Paracalyx and Flemingia did not have restriction site for this enzyme. Despite the analyses of four gene regions using 15 restriction enzymes, differentiation
at genus level could not be obtained. These observations indicated limited divergence of the chloroplast genome among the
four genera of the sub-tribe Cajaninae suggesting close relationships of the taxa.
Four DNA extraction protocols were compared for ability to produce DNA from the leaves or needles of several species: oak, elm, pine, fir, poplar and maize (fresh materials) and rhododendron (silica dried or frozen material). With the exception of maize and poplar, the species are known to be difficult for DNA extraction. Two protocols represented classical procedures for lysis and purification, and the other two were a combination of classical lysis followed by anion exchange chromatography. The DNA obtained from all procedures was quantified and tested by PCR and Southern hybridisation.Test results indicated superiority of one of the four protocols; a combination of CTAB lysis followed by anion exchange chromatography which enabled DNA extraction from all seven species. A second protocol also produced DNA from leaves or needles of all species investigated and was well suited for PCR applications but not Southern hybridisations. The remaining protocols produced DNA from some but not all species tested.Abbreviations: CTAB, hexadecyltrimethylammonium bromide; EtOH, Ethanol; TBE, tris-borate-EDTA.
Restriction fragment analysis of the chloroplast gene psbA was carried out in twelveAbies species native to Asia, Europe and North America using the four restriction enzymes. The pair of Asiatic representatives
ofA. koreana andA. veitchii differed profoundly not only from each other but also with respect to the European and Northamerican species. The variation
observed within the last mentioned groups of firs was mainly due to the different restriction profiles of the gene inA. alba andA. nordmanniana species of the former region as well as owing to its heterogenous nature in all the four species from North America. The
RFLP data presented to far only partially correlate with the taxonomic status and hybridological relationships of the species
concerned.
Keywords:gene arrangement;intraspecific polymorphism;intron;Quercus sp
The nucleotide sequence was obtained for the chloroplast gene coding for the large subunit of the ribulose 1,5-bisphosphate carboxylase (rbcL) of chestnut (Castanea sativa Mill.), a member of the woody family Fagaceae. Amplification primers downstream and upstream the rbcL open reading frame are also described. By comparing with other angiosperm sequences, we show that the rate of evolution of rbcL in the family Fagaceae is much slower than that observed for the families of annuals analyzed.Key words: angiosperms, Castanea sativa, Fagaceae, phylogeny, rbcL.
Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.
Restriction fragment analysis was conducted to determine interspecific chloroplast DNA (cpDNA) variation and genetic relationships among Populus deltoides, P. nigra, P. x canadensis (P. deltoides x P. nigra), and P. maximowiczii. Total cellular DNAs of these poplars were digested with 16 restriction endonucleases, and Southern blots of the restriction digests were probed with six different cloned cpDNA fragments from Petunia. P. deltoides, P. nigra, and P. maximowiczii each had a distinct chloroplast genome, separated by many restriction-site and restriction-fragment-length mutations, predominantly in the large single-copy region of the genome. P. x canadensis shared the same cpDNA restriction fragment patterns as P. deltoides var. deltoides. P. nigra was most diverged from P. deltoides, and P. deltoides showed close cpDNA relationships to P. maximowiczii. Nucleotide substitutions per site in cpDNA were 0.0036 between P. deltoides and P. maximowiczii, 0.0071 between P. nigra and P. maximowiczii, and 0.0077 between P. deltoides and P. nigra. We suggest that P. nigra should be classified in a new separate section, the Nigrae.
Representatives of seven genera from five tribes ofRubiaceae have been compared in respect to a non-coding intergene cpDNA region of about 1000 bp, situated between the atpB and the rbcL genes. The resulting most parsimonious PAUP cladogram corresponds very well with one based on total cpDNA restriction site data obtained byBremer & Jansen (1991). The two different molecular analyses thus corroborate each other and contribute to an improved systematic arrangement of the large family, e.g., in respect to placing the tribeHedyotideae clearly into the subfamilyRubioideae, closer toRubieae than toPsychotrieae.
A phylogenetic analysis of 25 species, representing eight genera of theRubieae tribe (Rubiaceae), has been made using the DNA sequence of the chloroplastatp B-rbc L intergene region. Six tropical genera from other tribes ofRubiaceae have been used as outgroups. Whatever the method of analysis (distance, parsimony or maximum likelihood), five groups are clearly separated and described as informal clades. Their relative relationships are not clearly resolved by the parsimony analysis, resulting in eight equally parsimonious trees, 327 steps long, with a consistency index (CI) of 0.749 (excluding uninformative sites). TheRubieae tribe appears monophyletic from the data available. Some new and partly unexpected phylogenetic relationships are suggested. The genusRubia forms a separate clade and appears to be the relatively advanced sister group of the remaining taxa. TheSherardia clade also includes the generaCrucianella andPhuopsis. Galium sect.Aparinoides appears closely attached to theAsperula sect.Glabella clade. The remaining taxa ofGalium are paraphyletic:Galium sect.Platygalium (in theCruciata clade) is linked to the advanced generaCruciata andValantia; the more apomorphic groups ofGalium form theGalium sect.Galium clade, including the perennial sectionsGalium, Leiogalium, andLeptogalium as well as the annual (and possibly polyphyletic) sect.Kolgyda.
The interrelationship of the ten species of the genusTyphonium and related genera in subtribe Arinae of the Araceae was inferred by chloroplast DNA restriction fragment analysis. A total
of 42 site mutations were observed and 26 site mutations were shared by two or more species. A majority rule consensus tree
was made by performing 100 bootstrap replicates using Wagner Parsimony. Two groups ofTyphonium were recognized significantly as monophyletic groups, i.e. 1)Typhonium larsenii andT. kunmingense, and 2)T. trilobatum, T. blumei andT. flagelliforme.
We have adapted a procedure for the isolation of genomic DNA from needles of silver fir (Abies alba) to meet the requirements for large-scale analysis of the population genetics of forest trees. Our modifications permit the
entire procedure to be carried out in Eppendorf tubes, which greatly minimizes time, plant material, and the amounts of chemicals.
DNA is recovered with a mean efficiency of 80 μg/g needles, is suitable for restriction by the common endonucleases, and serves
as a substrate for PCR.
Black pine chloroplast DNA is 119,707 bp long. The physical map is shown and the genes are listed. Plasmid clones covering
the entire DNA sequence have been ordered and available upon request.
Extensive variation in synonymous and nonsynonymous rates of substitution was observed among 50 sequences of the gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) representing bryophyte, conifer, dicot, and monocot taxa. Relative rate tests revealed rate differences of up to 138% for nonsynonymous substitutions and up to 85% for synonymous ones. Within angiosperms, the annual forms evolved more rapidly, on average, than perennial forms. This rate heterogeneity was more extensive at nonsynonymous sites than synonymous ones, and it resulted primarily from a recent acceleration of substitution rate in many groups of angiosperms.