Chloroplast DNA variation in Populus. I. Intraspecific restriction fragment diversity within Populus deltoides,P. nigra and P. maximowiczii

We examined intraspecific chloroplast (cp) DNA variation within Populus deltoides, P. nigra, and P. maximowiczii by restriction fragment analysis using 16 restriction endonucleases and six heterologous probes of cloned Petunia cpDNA fragments. All three Populus species showed intraspecific cpDNA variation, which was intra- and inter-varietal in P. deltoides, intervarietal in P. nigra, and origin-specific in P. maximowiczii. Two varieties of P. deltoides, var deltoides and var occidentalis, showed distinct cp genomes/DNA. Three distinct cp genomes/DNA, separated by a loss or gain of 1 EcoRV restriction site and/or 1 restriction fragment length polymorphism (RFLP), were observed among the individuals of P. deltoides var deltoides. Within P. nigra, cpDNA of var italica was distinct from that of vars nigra and plantierensis by one RFLP and by a loss or gain of one BamHI restriction site. Populus maximowiczii clones of Chinese origin were separated from those of Japanese origin by a gain or loss of one ClaI restriction site in their cpDNA. The estimate of nucleotide substitutions per site in cpDNA was 0.07% between two varieties of P. deltoides, 0.05% between var italica and var nigra or plantierensis of P. nigra, and 0.01% between Japanese and Chinese accessions of P. maximowiczii.     

Chloroplast DNA inheritance in Populus

Summary 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.     

Chloroplast DNA variation in Populus. III. Novel chloroplast DNA variants in natural Populus x canadensis hybrids

A rare phenomenon of the occurrence of novel non-parental chloroplast DNA (cpDNA) variants in natural sexual interspecific hybrids between Populus deltoides var deltoides and P. nigra, P. x canadensis is described. Restriction fragment variation of cpDNA in 17 P. x canadensis cultivars was examined and compared with that of representative samples of P. deltoides and P. nigra using 83 combinations of 16 restriction enzymes and six Petunia hybrida cpDNA probes. Twelve cultivars had one to five novel non-parental cpDNA fragments in the chloroplast genome region homologous to the 9.0-kb PstI cpDNA fragment of Petunia from the large single-copy region.     

Pollen competition among Populus deltoides Marsh., P. nigra L. and P. maximowiczii Henry in fertilizing P. deltoides ovules and siring its seed crop

Summary Interspecific pollen competition among Populus deltoides, P. nigra and P. maximowiczii in fertilizing P. deltoides ovules was studied by using a pollen mixture technique, allozymes and leaf morphology. The frequencies of F₁ seedlings of different paternities in pollen-mix crosses showed highly significant (P<0.01) departures from the 11 ratio expected if pollen selection was random. P. deltoides pollen was the most competitive. The mean percentages of F₁ seedlings of P. deltoides paternity in crosses with pollen mixes P. deltodes + P. nigra, P. deltoides + P. maximowiczii, and P. deltoides + P. nigra + P. maximowiczii were 95.0, 92.5, and 84.8, respectively. P. maximowiczii pollen was more competitive than P. nigra pollen, which was at a selective disadvantage. An average of 83.6% of F₁ progenies of the eight crosses with P. nigra + P. maximowiczii pollen showed P. maximowiczii paternity. Also, in four crosses with P. deltoides + P. nigra + P. maximowiczii pollen, the relative proportion of P. deltoides × P. maximowiczii seedlings (13.4%) was higher than that of P. deltoides × P. nigra seedlings (1.8%). Pollen proportions in the pollen mixes and pollen size did not significantly affect the competitive ability of the pollen. The relative pollen competitive ability indicated reproductive affinities among the species.     

Inheritance of chloroplast DNA inPopulus

Summary 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.     

Application of genetic markers to the discrimination of European Black Poplar (Populus nigra) from American Black Poplar (P. deltoides) and Hybrid Poplars (P. x canadensis) in Switzerland

European Black Poplar (Populus nigra) is considered a rare and endangered tree species because of severe reduction of its natural riverine habitat and potential hybridisation with the related non-indigenous taxa P. deltoides and P. x canadensis. As it is difficult to distinguish these taxa solely based on their morphology, we applied a PCR-based assay with an easy-to-use and robust molecular marker set (cpDNA trnL-trnF/RsaI RFLP, nDNA win3 and nDNA POPX/MspI RFLP) in order to identify pure P. nigra. Different plant tissues could be used for fast and standardised DNA extraction. The application of the three marker types was tested on a number of different Populus taxa, and they were also used for the verification of pure P. nigra in a sample of 304 putative P. nigra individuals from Switzerland. Cross-checking of the DNA data with those using a traditional allozyme approach resulted in complete agreement. The availability of molecular identification methods is an important prerequisite for the conservation of European Black Poplar, because pure, non-introgressed plant material can then be used in restoration projects of European floodplains.     

Natural hybridisation between <Emphasis Type="Italic">Populus nigra</Emphasis> L. and <Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">x canadensis</Emphasis> Moench. Hybrid offspring competes for niches along the Rhine river in the Netherlands

Black poplar (Populus nigra L.) is a major species for European riparian forests but its abundance has decreased over the decades due to human influences. For restoration of floodplain woodlands, the remaining black poplar stands may act as source population. A potential problem is that P. nigra and Populus deltoides have contributed to many interspecific hybrids, which have been planted in large numbers. As these Populus x canadensis clones have the possibility to intercross with wild P. nigra trees, their offspring could establish themselves along European rivers. In this study, we have sampled 44 poplar seedlings and young trees that occurred spontaneously along the Rhine river and its tributaries in the Netherlands. Along these rivers, only a few native P. nigra L. populations exist in combination with many planted cultivated P. x canadensis trees. By comparison to reference material from P. nigra, P. deltoides and P. x canadensis, species-specific AFLP bands and microsatellite alleles indicated that nearly half of the sampled trees were not pure P. nigra but progeny of natural hybridisation that had colonised the Rhine river banks. The posterior probability method as implemented in NewHybrids using microsatellite data was the superior method in establishing the most likely parentage. The results of this study indicate that offspring of hybrid cultivated poplars compete for the same ecological niche as native black poplars. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

A large scale survey of Populus nigra presence and genetic introgression from non-native poplars in Switzerland based on molecular identification

Populus nigra is considered a rare and threatened tree species in Switzerland because of dramatic habitat loss owing to river regulations during the last two centuries and because of potential gene introgression from non-indigenous P. deltoides through planted P. x canadensis hybrids. The significance of introgression as an endangerment to P. nigra, however, is controversial. The aims of the present study were (1) to assess how abundant P. nigra trees are in Switzerland and (2) to assess potential gene introgression. We present data from a molecular survey of 1372 putative P. nigra trees from Switzerland, using both chloroplast DNA and nuclear DNA markers. The results show that P. nigra is more abundant in Switzerland than hitherto thought. Furthermore, we detected a low frequency of gene introgression.     

Trinucleotide repeat microsatellite markers for black poplar (Populus nigra L.)

Using an enrichment procedure, we have cloned microsatellite repeats from black poplar (Populus nigra L.) and developed primers for microsatellite marker analysis. Ten primer pairs, mostly for trinucleotide repeats, produced polymorphic fragments in P. nigra. Some of them also showed amplification in other poplar species. (P. deltoides, P. tricocarpa, P. tremula, P. tremuloides, P. candicans, P. lasiocarpa). The best six loci were tested on 23 P. nigra genotypes collected across Europe. The microsatellites produced up to 12 alleles per locus in this set, with observed heterozygosity between 0.32 and 0.91.     

Diversity and evolution of chloroplast DNA in Triticum and Aegilops as revealed by restriction fragment analysis

Summary Restriction fragment analysis of chloroplast (cp) DNAs from 35 wheat (Triticum) and Aegilops species, including their 42 accessions, was carried out with the use of 13 restriction enzymes to clarify variation in their cpDNAs. Fourteen fragment size mutations (deletions/insertions) and 33 recognition site changes were detected among 209 restriction sites sampled. Based on these results, the 42 accessions of wheat-Aegilops could be classified into 16 chloroplast genome types. Most polyploids and their related diploids showed identical restriction fragment patterns, indicating the conservatism of the chloroplast genome during speciation, and maternal lineages of most polyploids were disclosed. This classification of cpDNAs was principally in agreement with that of the plasma types assigned according to phenotypes arising from nucleus-cytoplasm interactions. These mutations detected by restriction fragment analysis were mapped on the physical map of common wheat cpDNA, which was constructed with 13 restriction endonucleases. Length mutations were more frequently observed in some regions than in others: in a 16.0 kilo base pairs (kbp) of DNA region, including rbcL and petA genes, 6 of 14 length mutations were concentrated. This indicates that hot spot regions exist for deletions/insertions in chloroplast genome. On the other hand, 33 recognition site mutations seemed to be distributed equally throughout the genome, except in the inverted repeat region where only one recognition site change was observed. Base substitution rate (p) of cpDNA was similar to that of other plants, such as Brassica, pea and Lycopersicon, showing constant base substitution rates among related taxa and slow evolution of cpDNA compared with animal mitochondrial DNA. Phylogenetic relationships among Triticum and Aegilops species were discussed, based on the present data.Contributions no. 45 and no. 490 from the Kihara Institute for Biological Research, Yokohama City University and the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, respectively.     

Restriction fragment length polymorphisms of a chloroplast photosystem II gene from poplar and their use for species identification.

In a total DNA library from the poplar clone Beaupré (Populus trichocarpa x P. deltoides) one DNA clone was found to identify restriction site polymorphisms in different poplar species. This clone represents a cpDNA gene that shows close homology to a photosystem II gene of pea and spinach coding for the D2 protein and the 44 kDa reaction centre. In Southern blot analysis this probe identified interspecific restriction site variation among the different poplar species; intraspecific variation was not detectable. As the chloroplast genome is maternally inherited in poplars this cpDNA probe was used for identification of P. nigra or P. deltoides as the seed parents of F1 hybrid trees in natural stands of western Germany.     

Efficient Agrobacterium-mediated transformation of commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry

Many economically important species of Populus, especially those in sections Aigeiros and Tacamahaca, remain recalcitrant to genetic transformation. In this study, a simple and reliable protocol was developed for the efficient Agrobacterium-mediated transformation of a difficult-to-transform, but commercially viable, hybrid poplar Populus nigra L. × P. maximowiczii A. Henry (NM6). A plant transformation vector designed to express the β-glucuronidase (GUS) gene was used to detect transformation events at early stages of plant regeneration and to optimize parameters affecting poplar transformation. The use of zeatin riboside in shoot-induction medium, regeneration of shoots via indirect organogenesis, and early selection pressure were the major modifications that drastically improved the efficiency of poplar transformation and minimized the number of untransformed regenerants. Transgenic shoots were routinely obtained 4–10 weeks after co-culture with A. tumefaciens, with a greater than 90% rate of plant recovery. Stable transgene integration, ranging from a single insertion to ten copies per genome, was confirmed by Southern blot analysis. The mean transformation frequency was 36.3% and about two-thirds of the lines had 1–2 transgene copies. Among the explants, petioles and leaves had a higher transformation frequency than did stem segments. Growth characteristics and the morphology of transgenic poplar plants were identical to untransformed controls. These findings will accelerate the development of P. nigra × P. maximowiczii plants with novel traits, and may also be useful to improve transformation procedures for other Populus species.     

Chloroplast DNA differences between cultivated hop,Humulus lupulus and the related species H. japonicus

Chloroplast DNA (cpDNA) of Humulus Lupulus and H. japonicus was examined by restriction endonuclease analysis with BamHI, BanI, BclI, BstEII, DraI, EcoRI, EcoRV, HindIII, KpnI, PaeR7I, PstI, PvuII, SalI and XhoI. The restriction fragment patterns showed that the cpDNAs shared a large number of restriction sites. However, the chloroplast genomes of the two species could be distinguished by differences in restriction site and restriction fragment patterns in the PstI, PvuII, BclI, EcoRV, DraI and HindIII digests. On the basis of the complexity of restriction enzyme patterns, the enzymes PstI, PvuII, SalI, KpnI and XhoI were selected for mapping the chloroplast genomes. Single and double restriction enzyme digests of cpDNA from the two species were hybridized to cpDNA probes of barley and tobacco. The data obtained from molecular hybridization experiments were used to construct the cleavage site maps. Except for the PstI digest, the arrangement of cpDNA restriction sites was found to be the same for both species. An extra PstI site was present in H. lupulus. Three small insertions/deletions of about 0.8 kbp each were detected in the chloroplast genomes of the two species. Two of these insertions/deletions were present in the large and one in the small singlecopy region of the chloroplast genome. The cpDNA of Humulus was found to be a circular molecule of approximately 148 kbp that contains two inverted repeat regions of 23 kbp each, a small and a large single -copy region of approximately 20 kbp and 81 kbp, respectively. The chloroplast genome of hop has the same physical and structural organization as that found in most angiosperms.     

Dense genetic linkage maps of three Populus species (Populus deltoides, P. nigra and P. trichocarpa) based on AFLP and microsatellite markers

Populus deltoides, P. nigra, and P. trichocarpa are the most important species for poplar breeding programs worldwide. In addition, Populus has become a model for fundamental research on trees. Linkage maps were constructed for these three species by analyzing progeny of two controlled crosses sharing the same female parent, Populus deltoides cv. S9-2 x P. nigra cv. Ghoy and P. deltoides cv. S9-2 x P. trichocarpa cv. V24. The two-way pseudotestcross mapping strategy was used to construct the maps. Amplified fragment length polymorphism (AFLP) markers that segregated 1:1 were used to form the four parental maps. Microsatellites and sequence-tagged sites were used to align homoeologous groups between the maps and to merge linkage groups within the individual maps. Linkage analysis and alignment of the homoeologous groups resulted in 566 markers distributed over 19 groups for P. deltoides covering 86% of the genome, 339 markers distributed over 19 groups for P. trichocarpa covering 73%, and 369 markers distributed over 28 groups for P. nigra covering 61%. Several tests for randomness showed that the AFLP markers were randomly distributed over the genome.     

Evolutionary analysis of Pinus densata (Masters), a putative Tertiary hybrid.

Summary Restriction fragment analysis and heterologous hybridization of chloroplast (cp) DNA was used to develop species-specific markers for P. tabulaeformis, P. yunnanensis and P. massoniana. Fragment patterns created by the BclI and DraI restriction enzymes and hybridization patterns to the psbC and psbD probes were distinctive among the three species. No intraspecific variation was detected with respect to any of the cpDNA markers developed in this study. The cpDNA markers obtained were subsequently used to examine the parentage of P. densata, a putative Tertiary hybrid between P. tabulaeformis and P. yunnanensis. The analysis demonstrated for the first time that P. densata populations accommodate chloroplast genomes of P. tabulaeformis and P. yunnanensis, which strongly supports earlier suggestions of the hybrid origin of this species. It appears that P. densata represents a stabilized natural hybrid that has become adapted to high mountain environments where neither of the parental species can normally grow.     

Isolation and characterization of microsatellites in trembling aspen (Populus tremuloides)

 We have identified, isolated, and characterized microsatellite/simple sequence repeat (SSR) loci in trembling aspen (Populus tremuloides) by screening partial genomic libraries. We have also examined the compatibility and use of the P. tremuloides SSR primers to resolve microsatellites in other Populus species. Fourteen microsatellites were identified from 1600 clones screened. The TC/AG microsatellites were the most abundant. A total of 29 alleles were detected in 36 P. tremuloides individuals at the four SSR loci (two each of di- and tri-nucleotide repeats) characterized. The number of alleles at the SSR loci ranged from 5 to 11, with an average of 7.25 alleles per locus, and the observed heterozygosity ranged from 0.19 to 0.82, with a mean of 0.46 per locus. Although the highest polymorphism was observed for a dinucleotide SSR locus, the trinucleotide SSR loci showed substantial polymorphism. There were 34 unique multilocus genotypes among the 36 P. tremuloides individuals examined, and 89% of the individuals had unique multilocus genotypes. Two pairs of SSR primers were successful in PCR, amplifying genomic DNA and resolving microsatellites of comparable size from Populus deltoides, P. nigra, P.×canadensis, and P. maximowiczii. The microsatellite DNA markers developed could be used for clonal fingerprinting, certification of controlled crosses, genome mapping, marker-assisted early selection, genetic diversity assessments, and conservation and sustainable management of poplar genetic resources. Received: 14 November 1997 / Accepted: 17 November 1997     

Species‐specific microsatellite markers to monitor gene flow between exotic poplars and their natural relatives in eastern North America

Species‐specific microsatellite markers were obtained for the unambiguous recognition of five poplar species of ecological and commercial importance to eastern North America: the native species Populus balsamifera and Populus deltoides, the exotic species Populus maximowiczii, Populus nigra, Populus trichocarpa and their interspecific hybrids. Forty‐four of 71 tested primer pairs amplified simple sequence repeat (SSR) loci for all five taxa. Six of these loci showed non‐overlapping allelic diversity between species, including fixed differences. Together, they were useful to identify unambiguously the five taxa and to validate parental contributions in a group of hybrid progeny. These markers will be invaluable to detect gene flow from plantations of exotic poplar into adjacent stands of native species and between the two potentially hybridizing native species P. balsamifera and P. deltoides.     

Chloroplast DNA as an evolutionary marker in thePhaseolus vulgaris complex

We have analyzed the changes occurring in the chloroplast DNA (cpDNA) of taxa belonging to thePhaseolus vulgaris complex to help clarify relationships among species of this complex. Two restriction maps for 11 restriction enzymes comprising the whole chloroplast genome from a wildP. vulgaris and a wildP. coccineus accession were constructed. These maps allowed us to compare a total of 330 restriction sites between the two genomes in order to identify polymorphisms, assess the type of mutations detected, and identify regions of high variability. A region, located in the large single-copy region near the borders with the inverted repeats, accounted for a large portion of the variation. Most of the mutations detected were due to restriction sites gains or losses. Variable and conserved regions were then evaluated in 30 accessions belonging to taxa of theP. vulgaris complex. Phylogenetic analyses were made using parsimony methods. Conclusions obtained from such analyses were the following: (1) there was high cpDNA variability withinP. coccineus but not inP. vulgaris. (2)P. coccineus subsp.glabellus showed a very distinct cpDNA type that strongly suggests that it actually belongs to a different but as yet undetermined section of the genus. Our cpDNA observations are supported by distinctive morphological traits and reproductive biology of this taxon. (3) InP. coccineus subsp.darwinianus (also classified asP. polyanthus), the cpDNA lineage was in disagreement with data obtained from nuclear markers and suggested a reticulated origin by hybridization betweenP. coccineus as the male parent and an ancestralP. polyanthus type, closely allied toP. vulgaris, as the seed parent. This initial cross was presumably followed by repeated backcrossing toP. coccineus. Our cpDNA studies illustrate the importance of molecular markers in elucidating phylogenetic relationships. They also indicate that accurate phylogenies will require analyses of both nuclear and cytoplasmic genomes.     

Paternal plastid DNA can be inherited in lentil

Restriction fragment analysis was used to study the inheritance of chloroplast DNA (cpDNA) in F₁ progeny from crosses between Lens culinaris ssp. orientalis and L. culinaris ssp. culinaris. Twenty-five combinations of 11 restriction enzymes and three heterologous probes from Petunia hybrida cpDNA were used to screen six accessions of L.c. culinaris and one accession of L. c. orientalis for restriction fragment length polymorphisms (RFLPs). No variation in cpDNA was observed within the subspecies L. c. culinaris, but the L. c. orientalis accession was unambiguously distinguished from all six L. c. culinaris accessions by two RFLPs. Of ten F₁ progeny from L. c. orientalis x L. c. culinaris crosses, nine had only maternal cpDNA restriction fragments but one F₁ plant inherited cpDNA fragments from both parents. Nuclear DNA inheritance was biparental in all ten F₁ progeny.     

Chloroplast Genome of Vigna aconififolia and Localization of Certain Photosynthesis-related Genes

The restriction analysis of chloroplast genome of Vigna aeonitifolia has revealed that it is about 150 kb in size, similar to V. radiata. The restriction pattern of chloroplast DNA (cpDNA) for Pst I is also the same from both the species, but restriction fragment length polymorphism is observed in cases of Kpn I and Sstl. These differences in the restriction patterns have arisen because of the occurrence of different restriction sites in the chloroplast genome of V. aconitifolia. A restriction map of cpDNA for V. aeonitifolia has been prepared on the basis of these observations. Furthermore, seven genes (psbA, psbB, psbC, psbD, psaA, psaB and rbcL) — coding for polypeptides of photosystems I and II as well as the large subunit of ribulose 1,5-bisphosphate carboxylaseloxygenase — have been localized on the Pst I — and Kpn I — generated restriction fragments of V. aconitifolia with the help of heterologous gene-specific probes and their relative position on the restriction map is presented. The gene organization supports the view that an inversion of about 50 kb has occurred in Vigna cpDNA as compared to other species.