Microsatellites as DNA markers in Sitka spruce

Nine microsatellite loci were found by screening a genomic DNA library of Sitka spruce (Picea sitchensis) with the four oligonucleotide probes (TG), (CAC), (GATA) and (AT). Pairs of flanking primers were generated for seven microsatellites. Five primer pairs were used to screen up to 58 Sitka spruce clones. The five loci SStg3a, SStg4, SStg4a, SStg4c and SSgataS were found to have 15, 13, 4, 3 and 6 different length alleles respectively, and in using a combination of them almost all 58 Sitka spruce genotypes could be identified. The five primer pairs were successful in amplifying DNA from two other spruce species (Picea albutilia and Picea smithiana), while only one primer pair could amplify DNA from the pine species, Pinus sylvestris and Pinus latifolia. The inheritance of microsatellites in Sitka spruce was co-dominant Mendelian.     

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.     

Classifying seedlots of Picea sitchensis and P. glauca in zones of introgression using restriction analysis of chloroplast DNA

Summary Chloroplast DNA (cpDNA) restriction analysis was used to classify five reforestation seedlots as to species. The material included two Sitka spruce (Picea sitchensis (Bong.) Carr.), one white spruce (P. glauca (Moench) Voss) from interior British Columbia, and two putative hybrid seedlots from the coast-interior introgression zone in British Columbia. The cpDNA patterns generated by Bam-HI and Bc1-I from individual trees of Sitka spruce, white spruce, western white spruce (P. glauca var. albertiana (S. Brown)), and Engelmann spruce (P. engelmanni (Parry)) were species-specific. They were used as reference patterns for comparisons. In addition, two controlled crosses between white and Sitka spruce were analyzed to demonstrate the paternal inheritance of cpDNA in spruces. The cpDNA restriction patterns for the five seedlots were obtained from composite samples of seedlings from each lot and compared to the typical cpDNA patterns of each species. Restriction patterns for the two Sitka spruce seedlots agreed with those from the Sitka spruce tree, while patterns for the white spruce seedlots from British Columbia agreed with those from the white spruce tree, lacking evidence of any Engelmann spruce component in the sample. On the other hand, one putative hybrid seedlot showed cpDNA patterns similar to white spruce while the other showed fragments unique to both Sitka and white spruce, indicating that this was a hybrid seedlot. The analysis of cpDNA restriction polymorphism has proven to be an effective tool for classifying seedlots in regions of introgression. To our knowledge, these results provide the first demonstration of the use of cpDNA analysis for solving practical forestry problems.     

Inheritance of chloroplast and mitochondrial DNA in Picea and composition of hybrids from introgression zones

Summary The cloning of white spruce (Picea glauca) mitochondrial DNA homologous to the cytochrome oxidase II and ATPase genes of maize is described. These probes were used to define restriction fragment length polymorphisms which distinguish the white, Engelmann (P. engelmannii) and Sitka spruce (P. sitchensis) populations that occur in British Columbia. Analysis of progeny from crosses between the species revealed that mitochondrial DNA was maternally inherited in all cases (32 progeny from five independent crosses). The inheritance of chloroplast DNA was determined using a probe described previously; in this case, all progeny exhibited paternal inheritance (27 progeny from four crosses). Mitochondrial and chloroplast probes were used to test trees from zones of introgression between coastal (Sitka) and interior spruces (white and Engelmann). In most cases mitochondria and chloroplasts within individuals were contributed by different species. The data shows that there is a significant Sitka spruce component in trees east of the coastal watershed in British Columbia.     

Inheritance of plastids in interspecific hybrids of blue spruce and white spruce

Summary Chloroplast DNA (cpDNA) was purified from blue spruce (Picea pungens Engelm.) and white spruce [P. glauca (Moench) Voss], and was digested with several different restriction endonucleases. Restriction fragment length polymorphisms (RFLPs) were identified that differentiated the cpDNA of both species. Intraspecific conservation of the RFLPs that differentiated each species was confirmed by examining trees from across the natural range of each species. Ten F₁ hybrids were examined, and the cpDNA from each showed the banding pattern of the paternal species. Cloned Petunia cpDNA containing part of the rbcL gene hybridized to polymorphic bands, while a cloned maize mtDNA probe of the coxII gene failed to hybridize to any band.     

Genetic validation and characterization of RAPD markers differentiating black and red spruces: molecular certification of spruce trees and hybrids

 Picea mariana (black spruce) and P. rubens (red spruce) are closely related species which are difficult to differentiate morphologically. RAPD markers differentiating black and red spruces have been previously identified. In the present study, genetic validity of these markers was determined using samples representing range–wide provenances. Their applicability for certifying genetic identity of individual black, red trees and their hybrids from several sympatric and allopatric locations was demonstrated. These diagnostic fragments of both red and black spruce were present at a frequency of over 0.95 in allopatric provenances, but at a lower frequency in some sympatric provenances (0.43–1.00). Natural populations of red spruce exhibiting typical red spruce phenotype contained black spruce diagnostic RAPD fragments and black spruces growing in bogs with typical bog black spruce morphology, contained red spruce-specific RAPD markers. Some major RAPD markers were cloned and sequenced. The results reveal an extremely high degree of identity between the random primer and the primer binding sites on the genome. Amplification of black and red spruce genomic DNA with designed primers flanking the species-diagnostic RAPD markers indicates that most of RAPD markers used to differentiate black spruce from red spruce are not species specific since these sequences were detected in several spruce species using a more sensitive detection method. Received June 17, 2002; accepted August 5, 2002 Published online: February 4, 2003     

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.     

Karyotype of Norway spruce by multicolor FISH

The chromosomes (2n = 2x = 24) of Norway spruce are very large since their size reflects the huge amount of genomic DNA (2C = 30 × 10⁹ bp). However, the identification of homologous pairs is hampered by their high degree of similarity at the morphological level. Data so far presented in the literature were not sufficient to solve all the ambiguities in chromosome identification. Several genomic Norway spruce DNA clones containing highly repetitive sequences have been identified and characterised in our laboratory. Three of them were selected for fluorescent in situ hybridization (FISH) experiments because of their strong signals and suitability for chromosome identification: PATR140 hybridized at the centromeric site of three chromosome pairs; PAF1 hybridized in six subtelomeric and two centromeric sites; 1PABCD6 co-localized with the subtelomeric sites identified by PAF1. The statistical analysis of microscopic measurements of chromosomes in combination with the FISH signals of these probes allowed the unambigous construction of Norway spruce karyotype. We also compared the karyotype of Norway spruce with that of other spruce species to infer the number and kind of rearrangements that have occurred during the evolution of these species.Communicated by D.B. Neale     

Development of a set of highly polymorphic genomic microsatellites (gSSRs) in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Robust, polymorphic microsatellite DNA markers (simple sequence repeats—SSRs) are valuable tools for a range of tree conservation and breeding applications. SSRs are routinely used in the study of population genetic structure and diversity, pedigree reconstruction and genetic linkage mapping. Their abundance in the genome, co-dominant inheritance and potential for cross-species amplification make microsatellites highly prized markers. This paper characterises 22 novel genomic polymorphic microsatellite loci for Sitka spruce (Picea sitchensis (Bong.) Carr.). Amplification of DNA from Sitka spruce material was carried out both with a set of unrelated trees to obtain diversity statistics for each locus, and with the progeny of a full-sib family to test simple Mendelian inheritance. Observed heterozygosity ranged from 0.38 to 0.91 and allele number per locus ranged from 6 to 21, with a mean of 12.2. In addition, the primer pairs were tested with DNA from Norway spruce (P. abies) and white spruce (P. glauca) to investigate their potential for cross-species amplification and ten loci amplified in all three species. The results from these genomic microsatellites are compared to data generated from microsatellites derived from Picea EST libraries. In summary, this novel, highly polymorphic markers represent a significant addition to the rapidly expanding Picea genomics tool-box. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Homologues of the green algal gidA gene and the liverwort frxC gene are present on the chloroplast genomes of conifers

Strong hybridization signals were obtained from total DNA of two conifers, lodgepole pine (Pinus contorta) and Norway spruce (Picea abies), in a Southern blot analysis using a probe derived from the chloroplast gidA gene of the green alga Chlamydomonas reinhardtii. The pine fragments detected by the probe were found to originate from the chloroplast genome and, as judged by the signal intensity, this was also true for the spruce fragments. Sequence analysis of the hybridizing pine chloroplast DNA region revealed an open reading frame potentially encoding a 459 amino acid polypeptide, highly homologous to that deduced from the algal gene and to ORF465 of liverwort chloroplast DNA. Upstream of the gidA sequence, we found a trnN(GUU) gene and an open reading frame of 291 codons which was 78% identical to the frxC gene of liverwort. Since ORF465 is located immediately downstream of trnN and frxC in liverwort, the genetic organization of this region is very similar in the two plants. In contrast, neither the gidA nor the frxC gene is present in the chloroplast DNA of tobacco or rice. It was recently reported that deletions in the gidA region of the chloroplast genome of Chlamydomonas reinhardtii abolish the light-independent pathway of chlorophyll synthesis which exists in many algae and lower plants. The presence of the gidA gene on the chloroplast genomes of conifers may therefore be of significance with respect to the ability of these plants to synthesize chlorophyll in the dark.     

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.     

Triacylglycerols in the seeds of northern Scots pine,Pinus sylvestris L., and Norway spruce,Picea abies (L.) Karst.

The geographical variation in the composition of triacylglycerols in seeds of Scots pine (Pinus sylvestris L.) and Norway spruce [Picea abies (L.) Karst.], grown in Finland, was analyzed. The total lipid content of pine seeds was slightly higher in the northernmost provenance (68 °50N), whereas the lipid content of spruce seeds was not affected systematically by the geographical origin of the seeds. The species studied differed in the proportions of fatty acids in their triacylglycerols, though the three most abundant components were the same, i.e. oleic acid (181 n9), linoleic acid (182n-6) and 5,9,12-octadecatrienoic acid (183 5c9c12c). These fatty acids corresponded to more than 80 mol% of the total fatty acids. According to mass spectrometric analyses, the triacylglycerols of both spruce and pine seeds consisted of the same molecular species with 52–56 acyl carbons, but in different proportions. Molecules with 54 acyl carbons represented approximately 75% of the pine and 85% of the spruce triacylglycerols, with the most abundant molecular species being 545, 546 and 547. Some minor differences in the fatty acid composition of triacylglycerols of pine seeds from different seed collecting areas were found: the proportion of linoleic acid slightly increased whereas that of 5,11,14-eicosatrienoic acid decreased towards the northern origins. Similar differences were not found in the proportions of fatty acids in spruce seed lots. Furthermore, the proportions of triacylglycerols in both pine and spruce seeds from northern and southern collecting areas were not significantly different. The higher content of total lipids in spruce seeds compared with pine seeds may be due to the structure of the seed coat, and the lipophilic layers inside it, acting as a barrier to imbibition.     

拟南芥谷胱甘肽S-转移酶Zeta类进化酶的获得及其特性分析

拟南芥谷胱甘肽S-转移酶Zeta类(AtGSTZ)是一种与细胞代谢和环境净化密切相关的多功能酶.应用易错PCR和多轮DNA洗牌技术构建了AtGSTZ随机突变文库;再利用pH指示剂颜色改变法对突变文库进行筛选,获得了9个二氯乙酸脱氯活性提高的突变子.其中,NN23含25个氨基酸突变,比活力提高120%,NN20含24个氨基酸突变,比活力提高102%,EC1含2个氨基酸突变,比活力提高47%,其他6个为单点突变,比活力分别提高9%～60%.酶学分析显示,所有进化酶对底物二氯乙酸的催化效率和对谷胱甘肽的亲和力以及个别进化酶的复性能力都得到不同程度的提高,但热稳定性均没有明显改善.同时,对一系列与AtGSTZ空间折叠及催化活性相关位点进行了讨论.     

Structure of the intergenic spacer region from the ribosomal RNA gene family of white spruce (Picea glauca)

Five genomic clones containing ribosomal DNA repeats from the gymnosperm white spruce (Picea glauca) have been isolated and characterized by restriction enzyme analysis. No nucleotide variation or length variation was detected within the region encoding the ribosomal RNAs. Four clones which contained the intergenic spacer (IGS) region from different rDNA repeats were further characterized to reveal the sub-repeat structure within the IGS. The sub-repeats were unusually long, ranging from 540 to 990 bp but in all other respects the structure of the IGS was very similar to the organization of the IGS from wheat, Drosophila and Xenopus.     

The effects of host plant on the coccinellid functional response: Is the conifer specialist Aphidecta obliterata (L.) (Coleoptera: Coccinellidae) better adapted to spruce than the generalist Adalia bipunctata (L.) (Coleoptera: Coccinellidae)?

Female Aphidecta obliterata consumed an average of 26.6 ± 5.8 Elatobium abietinum and males consumed an average of 19.9 ± 3.2 Elatobium abietinum, but there was no significant difference in the number consumed between the sexes. In Petri dish trials, the larval stages of A. obliterata and all stages of Adalia bipunctata appeared to exhibit a Type II response to prey density, although A. obliterata adults showed a more linear response to prey density. There was no significant difference between the functional responses of the 3rd instars of the two coccinellid species, but there was a significant difference between the responses shown by the 1st instars, with A. obliterata larvae consuming more than those of A. bipunctata, especially at low densities, suggesting that the two species respond differently to an increase in prey density.At low prey densities, adults and 4th instars of both species exhibited a similar response to an increase in prey density but at higher densities the 4th instars and adult stages of A. bipunctata showed higher attack rates when compared with the same stages of A. obliterata.Adult and 4th instar A. obliterata exhibited Type II functional responses on spruce sections. The 4th instar A. obliterata larva appeared to be a more effective predator than the adult of the species, and was more effective when compared with adult A. bipunctata at lower prey densities but A. bipunctata adults appeared to be a more effective predator at higher prey densities.The host plant affected the rate of consumption by adult A. obliterata as adults on Sitka spruce sections consumed significantly higher numbers of aphids than their counterparts on Norway spruce. This was most noticeable at densities above 16 aphids.The distributions of the two coccinellid species in the olfactometer were significantly affected by the presence of host plant material. Aphidecta obliterata adults were found in significantly higher numbers in the Sitka spruce chambers than the control chambers (those without plant material). Adalia bipunctata adults were found in significantly lower numbers in the Norway spruce chamber than the control chamber.Although A. bipunctata has a higher level of voracity than A. obliterata, the latter is more adapted to the spruce environment and the boom and bust population dynamics of E. abietinum.     

Changes in biochemical composition of needles of ornamental dwarf spruce (Picea glauca ‘Conica’) induced by spruce spider mite (Oligonychus ununguis Jacobi, Acari: Tetranychidae) feeding

The impact of spruce spider mite (SSM) (Oligonychus ununguis Jacobi, Acari: Tetranychidae) feeding on needle compounds of young dwarf white spruce (Picea glauca ‘Conica’), important in defence against pests, was determined. It was shown that the direction and intensity of changes in chemical composition of spruce needles was related to the density of spruce spider mite population. Relative to uninfested controls, needles of trees infested by 2–6 mites per 5 cm oftwig during 8 weeks contained markedly higher concentration of soluble proteins, total phenolics and essential oil volatiles (linalool, β-phellandrene, β-myrcene, δ-3-carene, p-cymene, limonene, α-pinene, β-pinene, borneol, methyl salicylate, geranyl acetate). Feeding of a three times larger population of mites (18 specimens per 5 cm of twig) caused either reduction of concentration of those compounds, or no significant difference in comparison to the control. The results of our study show that O. ununguis at relatively low density (not exceeding 2–6 specimens per 5 cm of twig per 8 weeks) stimulates even susceptible host-plant to alter metabolism, however induced responses are suppressed as mite density increases. Findings are discussed in relation to the importance of changes in the concentration of needle primary and secondary metabolites to white spruce defence against O. ununguis.     

Abundance of two threatened woodpecker species in relation to the proportion of spruce plantations in native pine forests of western Norway

In a comparative study we investigated woodpecker abundance in forest landscapes with different proportion of native pine forest and spruce plantations in western Norway. In 100 circular study plots of 100ha each we recorded 38 white-backed –Dendrocopos leucotos, 22 grey-headed –Picus canus, 13 great spotted –Dendrocopos major, 6 green –Picus viridis, and 2 lesser spotted –Dendrocopos minor woodpeckers in the breeding season. The mean number of recorded woodpecker species peaked at 20–40% spruce plantations. The two most common species in the study, the white-backed and the grey-headed woodpeckers are both Red-listed species in Norway and among the rarest woodpeckers in Europe. The white-backed woodpecker preferred plots with higher than average proportions of standing dead trees and deciduous trees, and low proportions of spruce plantations in the plots. The grey-headed woodpecker preferred plots in the western (coastal) parts of the study area with presence of large aspen Populus tremula trees. Logistic regression models did not reveal any clear threshold values with respect to proportion of spruce plantations in plots, although both woodpecker species were extremely rare in plots with >60% spruce plantations. We recommend spruce plantations to be kept at moderate levels to ensure viable populations of woodpeckers in western Norway.     

Development of high‐density SNP genotyping arrays for white spruce (Picea glauca) and transferability to subtropical and nordic congeners

High‐density SNP genotyping arrays can be designed for any species given sufficient sequence information of high quality. Two high‐density SNP arrays relying on the Infinium iSelect technology (Illumina) were designed for use in the conifer white spruce (Picea glauca). One array contained 7338 segregating SNPs representative of 2814 genes of various molecular functional classes for main uses in genetic association and population genetics studies. The other one contained 9559 segregating SNPs representative of 9543 genes for main uses in population genetics, linkage mapping of the genome and genomic prediction. The SNPs assayed were discovered from various sources of gene resequencing data. SNPs predicted from high‐quality sequences derived from genomic DNA reached a genotyping success rate of 64.7%. Nonsingleton in silico SNPs (i.e. a sequence polymorphism present in at least two reads) predicted from expressed sequenced tags obtained with the Roche 454 technology and Illumina GAII analyser resulted in a similar genotyping success rate of 71.6% when the deepest alignment was used and the most favourable SNP probe per gene was selected. A variable proportion of these SNPs was shared by other nordic and subtropical spruce species from North America and Europe. The number of shared SNPs was inversely proportional to phylogenetic divergence and standing genetic variation in the recipient species, but positively related to allele frequency in P. glauca natural populations. These validated SNP resources should open up new avenues for population genetics and comparative genetic mapping at a genomic scale in spruce species.     

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     

Beauveria caledonica is a naturally occurring pathogen of forest beetles

In New Zealand, two introduced scolytid beetles, Hylastes ater and Hylurgus ligniperda (Curculionidae: Scolytinae) are pests in pine plantations. Investigation of the naturally occurring pathogens of these exotic pests revealed that both are attacked by Beauveria caledonica, a species originally isolated and described from soil in Scotland. The isolates in New Zealand were identical in morphology and conserved DNA region (rDNA, elongation factor α) sequence to isolates held in the USDA-ARS insect pathogens culture collection. In bioassay, the B. caledonica isolates were highly pathogenic to adults of H. ligniperda and larvae of Tenebrio molitor. Sporulation was observed on cadavers, confirming the species can utilise the cadavers. As both species were likely to have been introduced to New Zealand from Europe, a search was made for B. caledonica in the northern UK and Ireland. The fungus was found as a naturally-occurring pathogen of the weevil pest, Hylobius abietis (Curculionidae: Scolytinae), developing in spruce and other beetles in forests in both regions.