RAPD variations among pure and hybrid populations ofPicea mariana, P. rubens, andP. glauca (Pinaceae), and cytogenetic stability ofPicea hybrids: Identification of species-specific RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to determine genetic relationships amongP. mariana (black spruce),P. rubens (red spruce), andP. glauca (white spruce) and to assess the degree of polymorphism within populations from different provenances and among spruce hybrids. Eleven arbitrary decamer primers were used to amplify genomic DNAs extracted from embryogenic cultures and seedlings. Species-specific RAPD markers were identified.Picea mariana andP. rubens showed similar RAPD profiles confirming their close genetic relationship. Species-specific RAPD markers were identified and were useful in distinguishing white spruce from black and red spruces. RAPD differentiation between populations within each species was small. The level of polymorphism was much higher in spruce hybrid populations than in the pure species. Cytological analysis ofP. mariana ×P. rubens hybrids showed normal mitotic behaviour at prophase, metaphase, anaphase, and telophase. All the hybrids analyzed from different cross combinations were euploids.     

Species-specific nuclear and chloroplast single nucleotide polymorphisms to distinguish Picea glauca, P. mariana and P. rubens

 Picea rubens (red spruce) and P. mariana (black spruce) are closely related species which are difficult to differentiate morphologically. They are sympatric with P. glauca (white spruce) in the northern portion of their ranges. In order to identify potential interspecific polymorphisms, the chloroplast trnK intron and rpl33-psaJ-trnP region were sequenced, and the nuclear-encoded ITS region of the rDNA repeat was partially sequenced. Thirteen chloroplast and 12 nuclear candidate interspecific single nucleotide polymorphisms (SNPs) were identified. The species-specificity of several SNPs was determined by surveying DNAs amplified from trees representing range-wide provenance tests; these included 46 red spruce from 11 provenances, 84 black spruce from 30 provenances and 90 white spruce from 22 provenances. Two SNPs (1 chloroplast and 1 nuclear), which distinguish black spruce from red and white spruce, were consistent among 96–100% of the trees surveyed. Five SNPs (4 chloroplast and 1 nuclear), which distinguish white spruce from red and black spruce, were consistent among 100% of surveyed trees. These species-specific SNPs were used to identify anonymous spruce samples in a blind test, and their utility for small amounts of tissue, as little as single needles, was demonstrated. Scoring these SNPs is much less labor intensive than previous molecular methods for taxa differentiation (restriction fragment length polymorphisms or random amplified polymorphic DNAs), therefore they can be applied to large population studies. Received: 16 December 1998 / Accepted: 5 January 1999     

Species-specific RAPD fingerprints for the closely related Picea mariana and P. rubens

Species-specific molecular markers were designed to assist in the identification of closely related black spruce (Picea mariana [B.S.P.] Mill.) and red spruce (P. rubens Sarg.) in northeastern North America. Trees from six provenances of black spruce and three provenances of red spruce were sampled from outside the sympatric zone. They were first classified using a composite index of five qualitative morphological traits. The species-specific genetic markers were developed using random amplified polymorphic DNAs (RAPD) and a combination of bulk sample and individual tree analyses. Each species bulk sample was constructed from DNAs obtained from 12 trees that were from outside the sympatric zone and showed a morphological composite index specific of each species. A total of 161 primers were screened with the bulk samples. From these, 52 primers showing segregating fingerprints were further screened with the individual trees. Most of the markers observed were shared by the two species, and there was less diversity in P. rubens. A small number of markers were found to be monomorphic or nearly monomorphic and specific to either P. mariana or P. rubens. These markers remained species-specific when F₁ progenies derived from independent intraspecific crosses were screened, and they were subsequently found to co-segregate in hybrids derived from independent interspecific crosses here used as controls.     

Natural hybridization between black spruce and red spruce

Using species-specific random amplified polymorphic DNA (RAPD) markers and morphological characters, natural hybridization between the closely related black spruce Picea mariana (Mill.) B.S.P. and red spruce P. rubens Sarg. was evaluated in natural populations from north-eastern North America. Sampling included populations from both areas of allopatry and also 14 populations from part of the area of sympatry located in the province of Québec and covering several thousands of square kilometres. Classification results from RAPD species-specific markers and from a discriminant function based on morphology were compared. Molecular analysis of the allopatric populations indicated a small amount of interspecific gene leakage with no asymmetric directionality to introgression. A high occurrence of hybrid/introgressant individuals was observed within sympatric populations, suggesting weak reproductive isolation. As expected, the detection of such individuals was more efficient using molecular markers than with morphological traits. The hybrid zone appeared extensive with variable species structure and, in some stands, the main component composed of hybrid/introgressant trees. Implications for the genecology and genetic management of these species are discussed.     

Random Amplified Polymorphic DNA Analysis of Heterodera cruciferae and H. schachtii populations

Heterodera schachtii and H. cruciferae are sympatric in California and frequently occur in the same field upon the same host. We have investigated the use of polymerase chain reaction (PCR) amplification of nematode DNA sequences to differentiate H. schachtii and H. cruciferae and to assess genetic variability within each species. Single, random oligodeoxyribonucleotide primers were used to generate PCR-amplified fragments, termed RAPD (random amplified polymorphic DNA) markers, from genomic DNA of each species. Each of 19 different random primers yielded from 2 to 12 fragments whose size ranged from 200 to 1,500 bp. Reproducible differences in fragment patterns allowed differentiation of the two species with each primer. Similarities and differences among six different geographic populations of H. schachtii were detected. The potential application of RAPD analysis to relationships among nematode populations was assessed through cluster analysis of these six different populations, with 78 scorable markers from 10 different random primers. DNA from single cysts was successfully amplified, and genetic variability was revealed within geographic populations. The use of RAPD markers to assess genetic variability is a simple, reproducible technique that does not require radioisotopes. This powerful new technique can be used as a diagnostic tool and should have broad application in nematology.     

红皮云杉与嫩江云杉RAPD和ISSR分子标记反应体系优化和特异性检测

以红皮云杉和嫩江云杉为材料,采用现代分子标记技术-RAPD和ISSR标记研究红皮云杉与嫩江云杉间在基因组水平上的遗传差异。通过正交试验设计筛选RAPD和ISSR反应程序和反应体系。从近100个引物中筛选出3个RAPD引物和一个ISSR引物用来区分红皮云杉和嫩江云杉,它们分别是OPN07、OPA17、S25和ISSR67。用这4个引物扩增两种云杉基因组DNA,分别在1 000,950,1 500,2 000 bp处嫩江云杉有特异性谱带而红皮云杉没有。研究表明,采用RAPD和ISSR分子标记可以将红皮云杉和嫩江云杉在分子水平上加以区分,为今后进一步阐明云杉的系统进化,物种鉴定和种间杂交育种提供了理论依据。     

New evidence from mitochondrial DNA of a progenitor-derivative species relationship between black spruce and red spruce (Pinaceae)

Mitochondrial DNA (mtDNA) markers were used to assess the genetic diversity in allopatric populations of black spruce (Picea mariana [Mill.] BSP) and red spruce (P. rubens Sarg.). Patterns of mitochondrial haplotypes (mitotypes) were strikingly different between the two species. All mtDNA markers surveyed were polymorphic in black spruce, revealing four different mitotypes and high levels of mtDNA diversity (P(p) = 100%, A = 2.0, H = 0.496). In contrast, populations of red spruce had only two mitotypes and harbored low levels of ggenetic diversity (P(p) = 13.2%, A = 1.1, H = 0.120). When the southernmost allopatric populations of red spruce were considered, only one mitotype was detected. As previously reported for nuclear gene loci, the diversity observed for mtDNA in red spruce was a subset of that found in black spruce. Comparison of present and previously published data supports the hypothesis of a recent progenitor-derivative relationship between these species, red spruce presumably being derived by allopatric speciation of an isolated population of black spruce during the Pleistocene.     

Characterization of native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by PCR-RAPD based fingerprinting

Seventy isolates of Bacillus thuringiensis were isolated from soil samples collected from cotton fields. These isolates were characterized by randomly amplified poylmorphic DNA (RAPD) markers to determine their genetic diversity pattern based on their source of origin. Different random decamer primers were used for RAPD amplification, which generated a total of 1935 fragments; of these 1865 were polymorphic and 68 monomorphic. The primers OPA03, OPA08, OPD14, OPD19, OPD20, OPE17 and OPD19 produced 100% polymorphic fragments, whereas primers OPC06, OPC20 and OPD17 produced 20, 31 and 17 monomorphic fragments, respectively. When the RAPD banding pattern data was subjected to dendrogram construction, the 70 isolates fell into two separate clusters, cluster I and cluster II, which includes 26 and 44 B. thuringiensis isolates, respectively. These two main clusters were further divided into four subclusters at Eucledian distance of 150 and 80% similarity index. All primers showed amplification and indicated the good diversity of B. thuringiensis isolates. The RAPD pattern showed 4–10 bands per isolate, with MWt in the range of 0.4–3.5 Kb and an average of 193.5 fragments were produced per primer. The primer OPE17 was found to be the most discriminatory as it produced 286 polymorphic bands.     

Rapid identification of white-Engelmann spruce species by RAPD markers

Fragments of random amplified polymorphic DNA (RAPDs) were used as markers to distinguish Picea glauca (Moench) Voss (white spruce) and Picea engelmannii Parry (Engelmann spruce). These species and their putative hybrids are difficult to differentiate morphologically and are collectively known as interior spruce. Four oligodeoxynucleotide decamer primers showed species-specific amplification products between white spruce and Engelmann spruce. These fragments are highly conserved among seed lots and individual trees of each species from diverse geographic origins. The consistency and reproducibility of these species-specific amplification products were tested in more than two amplification reactions. Therefore, RAPD markers can provide genetic markers for easy and rapid identification of the specific genetic entry of these spruce species and their reported putative hybrids. According to the frequencies of the species-specific RAPD markers, it is possible to estimate the hybrid fraction, indicative of true introgression between the two species. These results are useful for quick identification of both species and their hybrid swarms at any stage in the sporophyte phase of the life cycle, for determining the occurrence and the magnitude of introgressive hybridization in an overlap zone between the two species, and for certification purposes in operational re-forestation and tree-improvement programs.     

Use of RAPD patterns for clone verification and in studying provenance relationships in Norway spruce (Picea abies)

 We have used the RAPD technique to analyse samples of Picea abies obtained from an improvement forestry station. Two types of plant material were harvested, the first being clones and the second provenances from various regions. We first checked the clonal identity of elite tree cuttings and clones; some differences in the RAPD patterns resulting from mis-planting or mis-labelling of cuttings were found. We also established a reference library of RAPD fingerprints for 96 clones, which will serve as a reference source in cases of litigation concerning clone identity. The RAPD technique was also used to study the genetic relationship between nine European provenances of Norway spruce. A dendogram was obtained by individual pairwise comparison of 42 RAPD bands, which separated the nine provenances into two major groups, one containing the Nordic provenances (Sweden and Bielorussia) and another the Alpine provenances (France, Austria, Germany and Belgium). The Belgian provenance, which is not indigenous, is most closely related to the German provenance. We conclude that the RAPD technique is a useful tool for forestry stations in managing propagation operations. Received: 15 June 1996 / Accepted: 11 October 1996     

Application of ISSR, RAPD, and cytological markers to the certification of Picea mariana, P. glauca, and P. engelmannii trees, and their putative hybrids.

Picea glauca (white spruce) and P. engelmannii (Engelmann spruce) are so similar and integrated that it is impossible to distinguish between them and their hybrids using morphological characteristics. Although natural hybrids between P. glauca and P. mariana (black spruce) do not generally occur, even though the 2 species are sympatric in North America, a first-generation hybrid, called the Rosendahl spruce, has been reported in the literature. In this study, several inter-simple sequence repeat (ISSR) markers were developed, as were randomly amplified polymorphic DNA (RAPD) markers, to certify spruce trees and their hybrids. ISSR fingerprinting was more efficient than RAPD assay; it detected 70% polymorphic DNA markers among the spruce species analyzed, whereas RAPD fingerprinting detected only 53%. Species-diagnostic ISSR and RAPD markers differentiating P. glauca from P. engelmannii and P. mariana were cloned and sequenced. Molecular certification of the spruce samples analyzed confirmed that all the seeds from interior spruce populations were true hybrids of P. glauca and P. engelmannii. But the analysis of seeds derived from the putative Rosendahl spruce indicated that this tree is likely a pure P. glauca genotype, rather than a hybrid of P. glauca and P. mariana. These data were confirmed by cytological analyses. Further analysis, using a more sensitive DNA amplification method with designed primers flanking the species-diagnostic ISSR and RAPD markers, revealed that such sequences are not generally species-specific because they are present in other spruce species.     

Food habit divergence between white-spotted charr and masu salmon in Japanese mountain streams: circumstantial evidence for competition

 The diet and foraging microhabitat of white-spotted charr, Salvelinus leucomaenis, were compared between mountain stream reaches where it occurs with (sympatric) and without (allopatric) masu salmon, Oncorhynchus masou masou, a potential competitor, to examine the evidence for interspecific competition between these fish, which commonly co-occur in Japanese mountain streams. In three streams examined, the similarity between the diets of allopatric charr and salmon was much greater than that between the diets of sympatric charr and salmon. Both allopatric charr and sympatric salmon intensively utilized terrestrial invertebrates among stream drifts (52%–65% of the diet), whereas this prey category made up only an intermediate portion of sympatric charr diets (11%–29%). Examination of available prey composition in stream drifts showed that the consumption of terrestrial invertebrates by allopatric charr and sympatric salmon was approximately twice as much as that by sympatric charr. The presence of salmon, a potential competitor, may alter the diet of charr in the sympatric reaches. Charr holding focal points closer to the streambed were considered less efficient than sympatric salmon in their utilization of terrestrial invertebrates drifting primarily on the stream surface, although the foraging microhabitat of the charr was not influenced by the salmon. The mechanisms responsible for the dietary divergence between sympatric charr and salmon are probably the consequence of scramble competition over terrestrial invertebrates drifting on the stream surface. Received: January 21, 2002 / Accepted: November 19, 2002 Acknowledgments We thank Y. Tokuda, T. Takasu, Y. Kaneda, H. Jyoya, and H. Aoe for their assistance. This work was partly supported by funding through the Takara Harmonist Fund by the Takara Syuzo Co. Ltd. and the Japan Ministry of Education, Science, Sport and Culture (grants 09NP1501 and 11440224). Correspondence to:H. Miyasaka     

Highly Informative Single-Copy Nuclear Microsatellite DNA Markers Developed Using an AFLP-SSR Approach in Black Spruce (Picea mariana) and Red Spruce (P. rubens)

Background

Microsatellites or simple sequence repeats (SSRs) are highly informative molecular markers for various biological studies in plants. In spruce (Picea) and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana) and red spruce (Picea rubens) using a simple but efficient method based on a combination of AFLP and microsatellite technologies.

Principal Findings

A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce and 30 red spruce individuals drawn from many populations. The number of alleles at a polymorphic locus ranged from 2 to 18, with a mean of 9.3 in black spruce, and from 3 to 15, with a mean of 6.2 alleles in red spruce. The polymorphic information content or expected heterozygosity ranged from 0.340 to 0.909 (mean = 0.67) in black spruce and from 0.161 to 0.851 (mean = 0.62) in red spruce. Ten SSR markers showing inter-parental polymorphism inherited in a single-locus Mendelian mode, with two cases of distorted segregation. Primer pairs for almost all polymorphic SSR loci resolved microsatellites of comparable size in Picea glauca, P. engelmannii, P. sitchensis, and P. abies.

Significance

The AFLP-based microsatellite-enriched library appears to be a rapid, cost-effective approach for isolating and developing single-locus informative genomic microsatellite markers in black spruce. The markers developed should be useful in black spruce, red spruce and other Picea species for various genetics, genomics, breeding, forensics, conservation studies and applications.     

Development of microsatellite markers for white spruce (Picea glauca) and related species

We report the development of 13 primer pairs that allow the unambiguous amplification of 15 microsatellite (SSR) loci in white spruce (Picea glauca). Fourteen of these loci were polymorphic in trees sampled at three geographically separated regions of western Canada. Segregation analysis carried out on these loci confirmed a Mendelian inheritance pattern for all except two, which showed significant segregation distortion. All of these primer pairs amplified SSR loci in at least one of the other Picea species tested [black spruce (P. mariana), red spruce (P. rubens), Norway spruce (P. abies), Colorado spruce (P. pungens), sitka spruce (P. sitchensis) and Engelmann spruce (P. engelmannii)]. Given the important commercial and ecological roles of these species, this set of markers will be invaluable for their management, the improvement of commercially important traits, and the study of their ecology and genetics. Received: 18 August 2000 / Accepted: 28 September 2000     

Identification of larch species (Larix decidua, Larix kaempferi and Larix X eurolepis) and estimation of hybrid fraction in seed lots by RAPD fingerprints

Species-specific RAPD markers were used to identify the different larch species (Larix decidua and Larix kaempferi) and their interspecific hybrid (Larix X eurolepis). Although morphological differences between pure species and the hybrids exist, differentiation is not always possible, especially at an early stage (seed or plantlet). Eleven RAPD markers differentiated the two larch species, and 4 species-specific markers were sufficient to estimate the F₁ hybrid fraction in a seed lot. The species-specific markers were tested on individual trees of European and Japanese larches of diverse geographic origins and on several seed lots of different origins (F₁, F₂ hybrids and pure species). The 4 specific markers found for the European larch and the Japanese larch were monomorphic and present in all provenances and in all F₁ hybrid trees tested. Polymorphic SCAR fragments were obtained for 3 of the 11 fragments originally selected for the RAPD screening phase. For 2 of them, the sequence had some homology with the mitochondrial genome of other organisms and is thus mitochondrial. The two mitochondrial fragments and the OPF-13₁₀₀₀ fragment exhibited one polymorphic band, thereby maintaining its species-specific identity: OPF-13₁₀₀₀ is specific to the European larch. The 4 RAPD primers selected in this study offer a reliable, quick and cheap tool for the identification of different larch species (Larix decidua and Larix kaempferi) and their interspecific hybrid (Larix X eurolepis). Received: 28 February 1999 / Accepted: 29 April 1999     

Contrasting Activity Patterns of Sympatric and Allopatric Black and Grizzly Bears

ABSTRACT The distribution of grizzly (Ursus arctos) and American black bears (U. americanus) overlaps in western North America. Few studies have detailed activity patterns where the species are sympatric and no studies contrasted patterns where populations are both sympatric and allopatric. We contrasted activity patterns for sympatric black and grizzly bears and for black bears allopatric to grizzly bears, how human influences altered patterns, and rates of grizzly-black bear predation. Activity patterns differed between black bear populations, with those sympatric to grizzly bears more day-active. Activity patterns of black bears allopatric with grizzly bears were similar to those of female grizzly bears; both were crepuscular and day-active. Male grizzly bears were crepuscular and night-active. Both species were more night-active and less day-active when ≤1 km from roads or developments. In our sympatric study area, 2 of 4 black bear mortalities were due to grizzly bear predation. Our results suggested patterns of activity that allowed for intra- and inter-species avoidance. National park management often results in convergence of locally high human densities in quality bear habitat. Our data provide additional understanding into how bears alter their activity patterns in response to other bears and humans and should help park managers minimize undesirable bear-human encounters when considering needs for temporal and spatial management of humans and human developments in bear habitats.     

Molecular differentiation of two vicariant neotropical tree species, Plathymenia foliolosa and P. reticulata (Mimosoideae), inferred using RAPD markers

 Plathymenia foliolosa and P. reticulata are two tropical tree species economically important for their high quality wood. P. foliolosa occurs mainly in areas of Atlantic Forest (a tropical forest) whereas P. reticulata occurs mainly in the Cerrado region (a savanna vegetation), both Brazilian biomes classified as hotspots for conservation priorities. In the present study RAPD markers were used to differentiate P. foliolosa from the vicariant species P. reticulata. Using 10 random primers it was possible to obtain 84 markers, of which 22 (26.2%) were present in just one of the two species. Nine of the ten primers were successful in discriminating between the species through the detection of diagnostic markers or band frequency differences. One of the populations of P. foliolosa, located in a region where the two species co-occur, presented several markers that were characteristic of P. reticulata, suggesting the occurrence of gene flow between the two species. Received September 3, 2001; accepted August 19, 2002 Published online: November 22, 2002 Addresses of the authors: Daniela Rodrigues Lacerda (dlacerda@dedalus.lcc.ufmg.br), Maria Bernadete Lovato (lovatomb@icb.ufmg.br), Maria Dolores Porto Acedo (acedomd@icb.ufmg.br), Dept. Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, CP: 486, Belo Horizonte, MG, 31270-901, Brasil. José Pires de Lemos Filho (lemos@icb.ufmg.br), Dept. Botanica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, CP: 486, Belo Horizonte, MG, 31270-901, Brasil.     

Genetic Relationship of <Emphasis Type="Italic">Curcuma</Emphasis> Species from Northeast India Using PCR-Based Markers

Molecular genetic fingerprints of nine Curcuma species from Northeast India were developed using PCR-based markers. The aim involves elucidating there intra- and inter-specific genetic diversity important for utilization, management, and conservation. Twelve random amplified polymorphic DNA (RAPD), 19 Inter simple sequence repeats (ISSRs), and four amplified fragment length polymorphism (AFLP) primers produced 266 polymorphic fragments. ISSR confirmed maximum polymorphism of 98.55% whereas RAPD and AFLP showed 93.22 and 97.27%, respectively. Marker index and polymorphic information content varied in the range of 8.64–48.1, 19.75–48.14, and 25–28 and 0.17–0.48, 0.19–0.48, and 0.25–0.29 for RAPD, ISSR, and AFLP markers, respectively. The average value of number of observed alleles, number of effective alleles, mean Nei’s gene diversity, and Shannon’s information index were 1.93–1.98, 1.37–1.62, 0.23–0.36, and 0.38–0.50, respectively, for three DNA markers used. Dendrograms based on three molecular data using unweighted pair group method with arithmetic mean (UPGMA) was congruent and classified the Curcuma species into two major clusters. Cophenetic correlation coefficient between dendrogram and original similarity matrix were significant for RAPD (r = 0.96), ISSR (r = 0.94), and AFLP (r = 0.97). Clustering was further supported by principle coordinate analysis. High genetic polymorphism documented is significant for conservation and further improvement of Curcuma species.     

Tracking the progression of speciation: variable patterns of introgression across the genome provide insights on the species delimitation between progenitor–derivative spruces (Picea mariana × P. rubens)

The genic species concept implies that while most of the genome can be exchanged somewhat freely between species through introgression, some genomic regions remain impermeable to interspecific gene flow. Hence, interspecific differences can be maintained despite ongoing gene exchange within contact zones. This study assessed the heterogeneous patterns of introgression at gene loci across the hybrid zone of an incipient progenitor–derivative species pair, Picea mariana (black spruce) and Picea rubens (red spruce). The spruce taxa likely diverged in geographic isolation during the Pleistocene and came into secondary contact during late Holocene. A total of 300 SNPs distributed across the 12 linkage groups (LG) of black spruce were genotyped for 385 individual trees from 33 populations distributed across the allopatric zone of each species and within the zone of sympatry. An integrative framework combining three population genomic approaches was used to scan the genomes, revealing heterogeneous patterns of introgression. A total of 23 SNPs scattered over 10 LG were considered impermeable to introgression and putatively under diverging selection. These loci revealed the existence of impermeable genomic regions forming the species boundary and are thus indicative of ongoing speciation between these two genetic lineages. Another 238 SNPs reflected selectively neutral diffusion across the porous species barrier. Finally, 39 highly permeable SNPs suggested ancestral polymorphism along with balancing selection. The heterogeneous patterns of introgression across the genome indicated that the speciation process between black spruce and red spruce is young and incomplete, albeit some interspecific differences are maintained, allowing ongoing species divergence even in sympatry. The approach developed in this study can be used to track the progression of ongoing speciation processes.