BACKGROUND
genetic maps provide an important genomic resource for understanding genome organization and evolution, comparative genomics, mapping genes and quantitative trait loci, and associating genes and genomic segments with phenotypic traits, especially in those species whose genomes are not yet completely sequenced.
for understanding the genetic architecture of species, genetic maps with high levels of genome coverage and confidence in the marker order are required.
high-density genetic maps and identification of genes or genetic factors controlling traits related to productivity, health, and adaptation to climatic change could accelerate forest tree improvement programs.
conifers are economically and ecologically important, and are the dominant tree species of the boreal and temperate forests.
genetic mapping and other genomics research is challenging in conifers, mainly because of their very large genome size   <cit> , the long time required to reach sexual maturity, inbreeding depression, and a general lack of advanced-generation pedigrees.
the genome of any conifer species has yet to be completely sequenced.
picea  is the second largest genus after pinus  in the family pinaceae of conifers.
black spruce  b.s.p.)
is a widespread transcontinental species of the north american boreal and temperate forests  <cit> , and has great ecological and economic importance.
it is one of the most important trees in canada for the production of pulp and paper, and is one of the most important reforestation species in canada  <cit> .
black spruce is a diploid species with haploid chromosome number  of  <dig> , like most other pinaceae members.
the estimated genome size of black spruce is large  with an approximate 2c genome length of about  <dig>  mpb http://www.rbgkew.org.uk/cval/homepage.html.
although the first genetic linkage map in conifers was constructed for a single white spruce  voss) tree in  <dig> from the analysis of haploid megagametophytes  <cit> , the progress in the spruce genome mapping has been rather slow, particularly compared with the genus pinus.
genetic linkage maps have been constructed for norway spruce   <cit> , white spruce  <cit> , and a black × red spruce  hybrid complex with an unknown proportion of the black spruce and red spruce genetic contribution to this hybrid  <cit> .
a parentage test with species-specific dna markers revealed that the crosses used in  <cit>  and  <cit>  harbored a substantial amount of the red spruce genetic background.
the markers used in the above-reviewed genome mapping studies were random amplified polymorphic dna  or a combination of rapd, amplified fragment length polymorphism , microsatellite/simple sequence repeat , expressed sequence tag polymorphism , selectively amplified microsatellite polymorphic loci , single nucleotide polymorphism , and/or  <dig> s rdna.
with the exception of the maps constructed for norway spruce and white spruce from f <dig> mapping populations  <cit>  and the map constructed for black × red spruce hybrids from f <dig> and bc <dig> mapping populations  <cit> , all other maps were constructed for single trees from the segregation of a small number of markers in haploid megagametophytes.
single-tree genetic maps are of limited value.
in predominantly outcrossing plants, such as conifers, a three-generation outbred pedigree  is considered to be more informative than f <dig> or bc <dig> pedigree  <cit> .
however, there is no published genetic linkage map in the genus picea based on a tgop.
moreover, most of the published spruce genetic linkage maps have not coalesced into  <dig> linkage groups corresponding to the haploid chromosome number of picea.
the linkage groups have ranged from  <dig> to  <dig>  the first single tree genetic linkage map of white spruce developed from  <dig> rapd markers coalesced into  <dig> linkage groups.
although the consensus map of picea abies  <cit>  and the composite map of p. mariana x p. rubens hybrids  <cit>  coalesced into  <dig> linkage groups, the maternal and/or paternal maps in these species coalesced into 13- <dig> linkage groups.
there is no information published on a genetic linkage map in pure black spruce.
the objective of this study was to construct a high-density genetic linkage map of black spruce.
we have used a three-generation outbred pedigree  to develop a high density to near-saturated genetic map of black spruce.
here, we report maternal, paternal, and near-saturated consensus genetic linkage maps developed for black spruce using aflp, sampl, estp, and ssr markers.
RESULTS
aflp markers
forty aflp primer combinations generated  <dig> markers segregating according to the expected mendelian ratios.
the number of polymorphic fragments ranged from  <dig> to  <dig>  with an average of  <dig> polymorphic fragments per primer combination .
the average number of polymorphic fragments obtained per primer combination was  <dig>   <dig>   <dig> with the use of three, four, and five selective nucleotides , respectively.
the size of the segregating polymorphic fragments without adapters ranged from  <dig> to  <dig> bp, with only one mapped fragment below  <dig> bp and seven between  <dig> and  <dig> bp.
of the  <dig> markers,  <dig> segregated in ratio of 1: <dig> and  <dig> in ratio of 3: <dig>  the number of markers segregating in the 1: <dig> ratio was  <dig> in the maternal parent and  <dig> in the paternal parent.
sampl markers
a total of  <dig> sampl markers, segregating according to the expected mendelian ratios, was obtained from the  <dig> sampl-msei primer combinations .
the fragment size without adapters ranged from  <dig> to  <dig> bp .
only six mapped sampl markers were of <  <dig> bp and  <dig> from  <dig> to  <dig> bp without adapters.
the number of polymorphic fragments ranged from  <dig> to  <dig>  with an average of  <dig> polymorphic fragments per sampl primer combination.
of the  <dig> sampl markers,  <dig> segregated in the ratio of 1: <dig>  whereas  <dig> sampl markers segregated in the ratio of 3: <dig>  the number of sampl markers segregating in the 1: <dig> ratio was  <dig> in the maternal parent and  <dig> in the paternal parent.
microsatellite markers
twenty white spruce est-based ssr loci were polymorphic between the parents of the mapping population and were mapped on the consensus map .
six of the  <dig> polymorphic loci were heterozygous in the female parent as dominant makers and fourteen in both parents as co-dominant makers.
two alleles at a ssr locus heterozygous in the male or female parent, segregated in a 1: <dig> ratio in the progeny.
where both the parents were heterozygous, the progeny segregated either in a 1:2: <dig> or 1:1:1: <dig> ratio for their parental alleles .
the known genes mapped using the microsatellites from white spruce est sequences were as follows: linkage group  i - cytochrome b <dig> ; lg iii - tir/p-loop/lrr ; lg vi - cytochrome p <dig>  and putative unc- <dig> ; lg vii - early light inducible protein  and chloroplast nucleoid dna binding protein ; lg viii - homeotic protein bel <dig> ; lg ix - auxin-induced protein  <dig> ; and lg xi- putative beta glycosidase  and gibberellin 12-oxidase .
six black spruce est-based ssr loci that were polymorphic between the parents as dominant markers were mapped on the consensus map; four were heterozygous in the female and two in the male parent for a null allele .
these markers segregated in a 1: <dig> ratio in the progeny.
sixteen black spruce genomic sequence-based ssr loci were polymorphic between the parents and were mapped on the consensus map .
eight microsatellite markers were heterozygous for a null allele in the female parent and five were heterozygous in the male parent.
these markers segregated in a 1: <dig> ratio as dominant markers.
three microsatellite loci were heterozygous in both parents and the progeny segregated in a 1:2: <dig> or 1:1:1: <dig> ratio as co-dominant markers.
estp markers
five estp markers showed mendelian segregation in the progeny.
three estp loci were heterozygous in the female parent and two were heterozygous in the male parent.
these markers segregated in a 1: <dig> ratio.
two estp markers were mapped on lg xi as well as one each on three linkage groups i, v, and ix .
genetic linkage maps
the maternal map consisted of  <dig> markers distributed on  <dig> linkage groups covering  <dig>  cm .
the number of mapped markers ranged from  <dig> to  <dig>  with an average of  <dig> markers per linkage group.
the length of the linkage groups ranged from  <dig> to  <dig> cm, with an average of  <dig> cm per linkage group .
the paternal map consisted of  <dig> markers assigned on  <dig> linkage groups, which covered  <dig>  cm .
the number of mapped markers ranged from  <dig> to  <dig>  with an average of  <dig> markers per linkage group.
the length of the linkage groups ranged from  <dig> to  <dig> cm, with an average of  <dig> cm per linkage group .
1within-parentheses are the numbers of inter-cross or co-dominant markers used for construction of the consensus maps.
the homologous linkage groups between the parents were identified on the basis of segregating intercross aflp, sampl, and ssr markers in the maternal and paternal maps.
at least three intercross markers per linkage group were used.
the integrated data set from the maternal and paternal maps allowed construction of a consensus linkage map.
the consensus linkage map composed of  <dig>  markers  mapped to  <dig> linkage groups .
the linkage groups correspond to the haploid chromosome number  of black spruce.
it is worth noting that we have consistently obtained  <dig> linkage groups for the maternal, paternal, and consensus linkage maps, unlike other mapping studies where parental and/or consensus maps did not coalesce into  <dig> linkage groups.
the consensus map covered  <dig>  cm, with an average of  <dig> markers per linkage group and an average of one marker every  <dig>  cm.
the size of linkage groups varied from  <dig> to  <dig> cm, with an average of  <dig> cm .
the maternal, paternal and consensus genetic maps for each of the  <dig> linkage groups aligned very well and showed almost perfect colinearity of the marker order .
genome length and map coverage
the estimated length of the black spruce genome was  <dig>  cm based on the method of chakravarti et al.
<cit> , and  <dig>  cm according to the method of fishmann et al.
<cit> .
the observed length of the black spruce genome obtained from the consensus genetic linkage map was  <dig>  cm.
thus, the consensus genetic map constructed in our study covered more than 97% of the estimated genome length of black spruce.
distribution of markers along linkage groups
significant deviations from the poisson distribution of markers were observed for marker intervals of  <dig>  cm,  <dig> cm,  <dig> cm,  <dig> cm, and  <dig> cm.
for a  <dig> cm interval, the significant deviation  is shown in figure  <dig>  indicating that the markers were not randomly distributed in the black spruce linkage groups.
marker distribution for other intervals  also showed clustering of markers  along linkage groups.
the independent analysis for testing the random distribution of aflp  and sampl  markers indicated deviations from the random distribution.
no correlation was observed between the number of mapped markers and the size of linkage groups.
these results further support the clustering of makers on the linkage map.
the distance between two adjacent markers on the linkage groups varied from  <dig> to  <dig>  cm, with an average distance of  <dig>  cm between any two adjacent markers .
this distance distribution reveals a strong skewness , further indicating the non-random distribution of the markers along the linkage groups .
among the  <dig>  intervals on  <dig> different linkage groups,  <dig> intervals were smaller than  <dig> cm , and  <dig> intervals were larger than  <dig> cm .
discussion
genetic linkage map
we have developed a high density to near-saturated and complete genetic linkage map of black spruce.
this is the first genetic map for black spruce, although genetic maps have recently been reported for a black spruce × red spruce hybrid complex  <cit> .
except in the southern part of its range, red spruce is largely sympatric with black spruce.
these two species hybridize in nature, although interspecific crossability represents a substantial but imperfect reproductive barrier for maintaining the separation of the species  <cit> .
the differentiation of black and red spruce and their interspecific hybrids based on dna markers, as used in pelgas et al.
<cit>  and pavy et al.
<cit> , is quite tenuous.
the parents of the mapping pedigree in our study represent pure black spruce  <cit> .
our black spruce genetic map is the first map in the genus picea based on a three-generation outbred pedigree .
there are only three other published genetic maps in the genus picea that are based on pedigreed material: one each in norway spruce and white spruce based on f <dig> mapping populations  <cit>  and one in the black × red spruce hybrids based on f <dig> and bc <dig> mapping populations  <cit> .
almost all other reported maps are for single trees .
the single-tree genetic maps are based on segregation of markers in haploid megagametophytes of maternal trees and do not take into account the segregation or recombination of markers in the paternal trees.
also, in conifers, the recombination rates were reported to be lower in female gametes than those in male gametes  <cit> .
thus, single-tree linkage maps are not as informative as genetic maps developed from diploid segregating pedigreed populations, and are specific to a single tree , and thus, having limited or often no use for qtl mapping.
black spruce, like most other spruce or pine species, is highly outbred  <cit> .
for genome and qtl mapping in outbred plants, tgop is more informative than any other pedigree used so far in the genus picea  <cit> .
indeed, tgop not only allows differentiation of up to four segregating alleles at a locus but also establishment of linkage phase among alleles in the mapping population  <cit> .
this information is required to use genetic linkage maps for qtl mapping.
furthermore, the outbred pedigrees are more representative of natural populations in an outbred plant.
in pinus, where the genome and qtl mapping work has been more advanced than in its sister genus picea, high-density genetic and qtl maps have been prepared using tgop in loblolly  and maritime  pine  <cit> .
*minor linkage groups
genome length and map coverage
the black spruce genome length estimated in our study is  <dig>  cm .
this is comparable with the genome length of  <dig>  or  <dig>  cm  reported for black × red spruce hybrids based on a composite map of f <dig> and/or bc <dig> mapping populations   <cit>  and that of  <dig>  cm estimated for black × red spruce controlled cross hybrids from a bc <dig> mapping population .
these results are consistent with the length of the genetic map for black spruce that is likely to be between  <dig> and  <dig> cm.
the length of the black spruce genetic map observed in our study is about 15% shorter than that reported for norway spruce   <cit>  and white spruce   <cit>  maps.
the genome size  of norway spruce   <cit>  and white spruce   <cit>  is  <dig> % and  <dig> %, respectively, higher than that of black spruce   <cit> .
although no direct relationships between the nuclear dna contents and genetic map lengths apparently exist, the shorter genetic map length in black spruce than in norway spruce or white spruce is apparently consistent with its comparatively smaller genome size.
our results suggest that the genome length observed in our study covers more than 97% of the estimated black spruce genome length.
thus, the black spruce genetic map reported here can very well be considered as almost complete.
this is the highest map coverage so far for any picea species.
the estimated length and the extent of coverage of genetic maps in different species could vary owing to differences in the mapping populations used, variation in recombination rates of the parents, and the number and types of markers used in linkage map construction  <cit> .
the mapping pedigree and markers used in our study are different from those used in norway spruce and white spruce .
linkage groups and marker density
in our study, the paternal, maternal, and consensus linkage maps consistently coalesced into  <dig> linkage groups, corresponding to the haploid chromosome number  in black spruce.
by contrast, in all other studies reported on genome mapping in the genus picea , with one exception, maternal, paternal, and/or consensus map did not coalesce into  <dig> linkage groups .
the consensus map reported here has  <dig>  markers distributed over  <dig> linkage groups, which represents an almost complete coverage of the black spruce genome, as the number of linkage groups corresponds to the haploid chromosome number.
the average distance observed among adjacent markers mapped for the genetic map of black spruce in our study  is comparable with or lower than that reported for the composite map of black spruce × red spruce complex  <cit> , as well as lower than that reported for norway spruce  .
however, the marker density of the black spruce genetic map reported here is the highest for any genetic map based on a single cross in the genus picea, as well for the maternal and paternal genetic maps .
marker systems
the genetic map of black spruce was constructed using aflp, sampl, ssr, and estp marker systems.
the aflp and sampl systems provided a sufficient number of anonymous polymorphic and informative markers to construct a high density to near-saturated genetic map, whereas the ssr and estp systems provided highly informative and co-dominant markers.
although ssr and estp markers, due primarily to their codominant nature, would be preferred for genome mapping, availability of limited numbers of these markers precludes constructing a high density to saturated genetic map in conifers using only these markers.
we mapped  <dig> aflp markers that were resolved by  <dig> primer combination, showing an average multiplex ratio of  <dig> markers per primer combination.
this multiplex ratio is comparable to that observed in norway spruce   <cit>  and loblolly pine   <cit> .
we mapped  <dig> sampl markers on  <dig> different linkage groups.
the multiplex ratio for sampl markers  was comparable to that observed for aflp markers.
the only other report where sampl markers have been used for genetic linkage mapping in conifers is for norway spruce  <cit> , where  <dig> sampl markers were mapped using two primer combinations.
a large number of aflp and sampl markers segregated in the 3: <dig> ratio, which suggests that the parental genomes are highly heterozygous.
as aflp and sampl markers were dominant, the 3: <dig> segregating  markers were useful for aligning the parental maps to construct the consensus map, which cannot be established directly.
also the intercross markers can help to identify additional linkage groups that were not represented in the parental maps  <cit> .
ssr loci provided highly informative markers.
forty-two ssr loci were mapped onto  <dig> linkage groups;  <dig> of these were highly informative for integrating the maternal and paternal maps to construct the consensus map.
the mapped cdna-based ssr markers are excellent candidates for comparative and composite mapping because these markers are expected to show high intraspecific homology and high interspecific orthology .
also these markers allowed mapping of  <dig> known genes on seven different linkage groups.
most est primer pairs resolved multilocus patterns, which is not surprising given the occurrence of multigene families in conifers  <cit> .
also, the rate of polymorphism observed in estps was very low, which suggests that more powerful methods, such as detection of single nucleotide polymorphisms  can be used to increase the resolution of polymorphism  <cit> .
nevertheless, the mapped estp loci along with cdna-based ssr markers provide good candidates for comparative mapping in picea, pinaceae, or other conifers  <cit> .
the genetic map presented here is the first-generation genetic map for black spruce, which provides a framework to map snp and other markers in the second-generation genetic map.
we are planning to map snp markers from candidate genes and genes expressed differentially in response to climate change and snps mapped by pavy et al.
<cit>  onto this map in future.
this is further discussed below under the future perspective section.
the mapped snp markers from expressed genes are quite useful for qtl and association mapping of relevant traits because allelic variation in the genes could be linked or associated with trait phenotypes.
however, these markers represent a very small proportion of the spruce genome.
assuming  <dig>  expressed genes of average  <dig> kb size and genome size of  <dig> gbp in black spruce, the transcribed genome represents less than  <dig> % of the whole genome.
snps in about  <dig> candidate genes are normally mapped, further reducing the proportion of the genome sampled to less than  <dig> %, although the mapped genes may have genome-wide distribution.
the anonymous markers, such as aflp and sampl markers generally provide a whole-genome scan; thus a genetic map using these markers may cover a large proportion of the genome, if not the whole genome.
however, anonymous markers such as aflp and sampl, have limitations in tagging genes controlling traits of interest via qtl and association mapping because variation in these markers may not represent functional genetic variation.
nevertheless, the black spruce genetic map reported here provides a valuable genomic resource in picea and pinaceae.
clustering of markers
even though only those markers segregating in mendelian ratios and not those showing a distorted segregation were used for the linkage analysis, clustering of aflp and sampl markers was detected in the linkage groups of black spruce.
these results agree with the clustering of aflp markers reported for genetic maps of picea abies  <cit> , pinus taeda  <cit> , and pinus sylvestrisl <cit> , but in contrast to random distribution of aflp markers in the genetic maps reported for norway spruce  <cit>  and black × red spruce hybrids  <cit> .
it should be noted that the study of scotti et al.
<cit>  was specifically performed to examine the distribution of marker classes in a genetic linkage map of norway spruce.
the non-random distribution of markers may be caused by non-random and unequal crossing over and recombination along the chromosome length.
the recombination is suppressed in the centromeric and heterochromatic pericentromeric regions  <cit> , and the presence of heterochromatin in pericentromeric regions is a general feature of plant chromosomes.
assuming a random distribution of markers, low levels of meiotic recombination may well cause markers that are physically well separated, to cluster on a linkage map.
future perspectives
the genetic map reported here is suitable for constructing a composite map of two tgop in black spruce and for the envisaged comparative mapping with red spruce and black spruce × red spruce hybrid.
the mapped est-based microsatellites and estps will provide very useful markers for this work.
we are also mapping additional est-based ssr markers.
black spruce and red spruce are highly genetically related species but their evolutionary relationships are controversial.
comparative genome mapping may shed some light on comparative genome organization  and evolution of these species.
the map could also be used for comparative mapping with other genera of pinaceae.
we are genotyping and phenotyping the larger mapping populations of the tgop used in this study and another tgop  of black spruce for qtl mapping of traits related to growth and adaptation to climate change.
we plan to use about  <dig>  snps from candidate genes and genes expressed differentially in response to climate change conditions as well as snps mapped by pavy et al.
<cit>  for our qtl mapping work, using a high throughput snp genotyping platform such as illumina's golden gate genotyping assay http://www.illumina.com/technology/goldengate_genotyping_assay.ilmn.
thus, the technologies, markers and genetic map developed in the present study provide an invaluable genomic resource for basic and applied genomics studies.
CONCLUSIONS
we have constructed the first high density to near-saturated genetic linkage map of black spruce, with above 97% genome coverage.
the maternal, paternal, and consensus maps in our study consistently coalesced into  <dig> linkage groups, corresponding to the haploid chromosome number  of  <dig> in the genus picea.
also, this is the first genetic map based on a three-generation outbred pedigree in the genus picea.
the genome length in p. mariana is likely to be about  <dig>  cm.
the genetic maps developed in our study can serve as a reference map for various genomics studies and applications in picea and pinaceae.
it will provide a foundation and a valuable resource for comparative mapping, constructing composite maps, and mapping quantitative trait loci and determining the genetic basis of complex quantitative traits of interest, such as growth and adaptation to climate change.

 genetic maps provide an important genomic resource for understanding genome organization and evolution, comparative genomics, mapping genes and quantitative trait loci, and associating genes and genomic segments with phenotypic traits, especially in those species whose genomes are not yet completely sequenced. for understanding the genetic architecture of species, genetic maps with high levels of genome coverage and confidence in the marker order are required. high-density genetic maps and identification of genes or genetic factors controlling traits related to productivity, health, and adaptation to climatic change could accelerate forest tree improvement programs. conifers are economically and ecologically important, and are the dominant tree species of the boreal and temperate forests. genetic mapping and other genomics research is challenging in conifers, mainly because of their very large genome size, the long time required to reach sexual maturity, inbreeding depression, and a general lack of advanced-generation pedigrees. the genome of any conifer species has yet to be completely sequenced. picea is the second largest genus after pinus in the family pinaceae of conifers. black spruce b.s.p.) is a widespread transcontinental species of the north american boreal and temperate forests, and has great ecological and economic importance. it is one of the most important trees in canada for the production of pulp and paper, and is one of the most important reforestation species in canada. black spruce is a diploid species with haploid chromosome number of, like most other pinaceae members. the estimated genome size of black spruce is large with an approximate 2c genome length of about mpb http://www.rbgkew.org.uk/cval/homepage.html. although the first genetic linkage map in conifers was constructed for a single white spruce voss) tree in from the analysis of haploid megagametophytes, the progress in the spruce genome mapping has been rather slow, particularly compared with the genus pinus. genetic linkage maps have been constructed for norway spruce, white spruce, and a black × red spruce hybrid complex with an unknown proportion of the black spruce and red spruce genetic contribution to this hybrid. a parentage test with species-specific dna markers revealed that the crosses used in and harbored a substantial amount of the red spruce genetic background. the markers used in the above-reviewed genome mapping studies were random amplified polymorphic dna or a combination of rapd, amplified fragment length polymorphism, microsatellite/simple sequence repeat, expressed sequence tag polymorphism, selectively amplified microsatellite polymorphic loci, single nucleotide polymorphism, and/or s rdna. with the exception of the maps constructed for norway spruce and white spruce from f mapping populations and the map constructed for black × red spruce hybrids from f and bc mapping populations, all other maps were constructed for single trees from the segregation of a small number of markers in haploid megagametophytes. single-tree genetic maps are of limited value. in predominantly outcrossing plants, such as conifers, a three-generation outbred pedigree is considered to be more informative than f or bc pedigree. however, there is no published genetic linkage map in the genus picea based on a tgop. moreover, most of the published spruce genetic linkage maps have not coalesced into linkage groups corresponding to the haploid chromosome number of picea. the linkage groups have ranged from to the first single tree genetic linkage map of white spruce developed from rapd markers coalesced into linkage groups. although the consensus map of picea abies and the composite map of p. mariana x p. rubens hybrids coalesced into linkage groups, the maternal and/or paternal maps in these species coalesced into 13- linkage groups. there is no information published on a genetic linkage map in pure black spruce. the objective of this study was to construct a high-density genetic linkage map of black spruce. we have used a three-generation outbred pedigree to develop a high density to near-saturated genetic map of black spruce. here, we report maternal, paternal, and near-saturated consensus genetic linkage maps developed for black spruce using aflp, sampl, estp, and ssr markers.  aflp markers forty aflp primer combinations generated markers segregating according to the expected mendelian ratios. the number of polymorphic fragments ranged from to with an average of polymorphic fragments per primer combination. the average number of polymorphic fragments obtained per primer combination was with the use of three, four, and five selective nucleotides, respectively. the size of the segregating polymorphic fragments without adapters ranged from to bp, with only one mapped fragment below bp and seven between and bp. of the markers, segregated in ratio of 1: and in ratio of 3: the number of markers segregating in the 1: ratio was in the maternal parent and in the paternal parent. sampl markers a total of sampl markers, segregating according to the expected mendelian ratios, was obtained from the sampl-msei primer combinations. the fragment size without adapters ranged from to bp. only six mapped sampl markers were of < bp and from to bp without adapters. the number of polymorphic fragments ranged from to with an average of polymorphic fragments per sampl primer combination. of the sampl markers, segregated in the ratio of 1: whereas sampl markers segregated in the ratio of 3: the number of sampl markers segregating in the 1: ratio was in the maternal parent and in the paternal parent. microsatellite markers twenty white spruce est-based ssr loci were polymorphic between the parents of the mapping population and were mapped on the consensus map. six of the polymorphic loci were heterozygous in the female parent as dominant makers and fourteen in both parents as co-dominant makers. two alleles at a ssr locus heterozygous in the male or female parent, segregated in a 1: ratio in the progeny. where both the parents were heterozygous, the progeny segregated either in a 1:2: or 1:1:1: ratio for their parental alleles. the known genes mapped using the microsatellites from white spruce est sequences were as follows: linkage group i - cytochrome b; lg iii - tir/p-loop/lrr; lg vi - cytochrome p and putative unc-; lg vii - early light inducible protein and chloroplast nucleoid dna binding protein; lg viii - homeotic protein bel; lg ix - auxin-induced protein; and lg xi- putative beta glycosidase and gibberellin 12-oxidase. six black spruce est-based ssr loci that were polymorphic between the parents as dominant markers were mapped on the consensus map; four were heterozygous in the female and two in the male parent for a null allele. these markers segregated in a 1: ratio in the progeny. sixteen black spruce genomic sequence-based ssr loci were polymorphic between the parents and were mapped on the consensus map. eight microsatellite markers were heterozygous for a null allele in the female parent and five were heterozygous in the male parent. these markers segregated in a 1: ratio as dominant markers. three microsatellite loci were heterozygous in both parents and the progeny segregated in a 1:2: or 1:1:1: ratio as co-dominant markers. estp markers five estp markers showed mendelian segregation in the progeny. three estp loci were heterozygous in the female parent and two were heterozygous in the male parent. these markers segregated in a 1: ratio. two estp markers were mapped on lg xi as well as one each on three linkage groups i, v, and ix. genetic linkage maps the maternal map consisted of markers distributed on linkage groups covering cm. the number of mapped markers ranged from to with an average of markers per linkage group. the length of the linkage groups ranged from to cm, with an average of cm per linkage group. the paternal map consisted of markers assigned on linkage groups, which covered cm. the number of mapped markers ranged from to with an average of markers per linkage group. the length of the linkage groups ranged from to cm, with an average of cm per linkage group. 1within-parentheses are the numbers of inter-cross or co-dominant markers used for construction of the consensus maps. the homologous linkage groups between the parents were identified on the basis of segregating intercross aflp, sampl, and ssr markers in the maternal and paternal maps. at least three intercross markers per linkage group were used. the integrated data set from the maternal and paternal maps allowed construction of a consensus linkage map. the consensus linkage map composed of markers mapped to linkage groups. the linkage groups correspond to the haploid chromosome number of black spruce. it is worth noting that we have consistently obtained linkage groups for the maternal, paternal, and consensus linkage maps, unlike other mapping studies where parental and/or consensus maps did not coalesce into linkage groups. the consensus map covered cm, with an average of markers per linkage group and an average of one marker every cm. the size of linkage groups varied from to cm, with an average of cm. the maternal, paternal and consensus genetic maps for each of the linkage groups aligned very well and showed almost perfect colinearity of the marker order. genome length and map coverage the estimated length of the black spruce genome was cm based on the method of chakravarti et al., and cm according to the method of fishmann et al.. the observed length of the black spruce genome obtained from the consensus genetic linkage map was cm. thus, the consensus genetic map constructed in our study covered more than 97% of the estimated genome length of black spruce. distribution of markers along linkage groups significant deviations from the poisson distribution of markers were observed for marker intervals of cm, cm, cm, cm, and cm. for a cm interval, the significant deviation is shown in figure indicating that the markers were not randomly distributed in the black spruce linkage groups. marker distribution for other intervals also showed clustering of markers along linkage groups. the independent analysis for testing the random distribution of aflp and sampl markers indicated deviations from the random distribution. no correlation was observed between the number of mapped markers and the size of linkage groups. these results further support the clustering of makers on the linkage map. the distance between two adjacent markers on the linkage groups varied from to cm, with an average distance of cm between any two adjacent markers. this distance distribution reveals a strong skewness, further indicating the non-random distribution of the markers along the linkage groups. among the intervals on different linkage groups, intervals were smaller than cm, and intervals were larger than cm. discussion genetic linkage map we have developed a high density to near-saturated and complete genetic linkage map of black spruce. this is the first genetic map for black spruce, although genetic maps have recently been reported for a black spruce × red spruce hybrid complex. except in the southern part of its range, red spruce is largely sympatric with black spruce. these two species hybridize in nature, although interspecific crossability represents a substantial but imperfect reproductive barrier for maintaining the separation of the species. the differentiation of black and red spruce and their interspecific hybrids based on dna markers, as used in pelgas et al. and pavy et al., is quite tenuous. the parents of the mapping pedigree in our study represent pure black spruce. our black spruce genetic map is the first map in the genus picea based on a three-generation outbred pedigree. there are only three other published genetic maps in the genus picea that are based on pedigreed material: one each in norway spruce and white spruce based on f mapping populations and one in the black × red spruce hybrids based on f and bc mapping populations. almost all other reported maps are for single trees. the single-tree genetic maps are based on segregation of markers in haploid megagametophytes of maternal trees and do not take into account the segregation or recombination of markers in the paternal trees. also, in conifers, the recombination rates were reported to be lower in female gametes than those in male gametes. thus, single-tree linkage maps are not as informative as genetic maps developed from diploid segregating pedigreed populations, and are specific to a single tree, and thus, having limited or often no use for qtl mapping. black spruce, like most other spruce or pine species, is highly outbred. for genome and qtl mapping in outbred plants, tgop is more informative than any other pedigree used so far in the genus picea. indeed, tgop not only allows differentiation of up to four segregating alleles at a locus but also establishment of linkage phase among alleles in the mapping population. this information is required to use genetic linkage maps for qtl mapping. furthermore, the outbred pedigrees are more representative of natural populations in an outbred plant. in pinus, where the genome and qtl mapping work has been more advanced than in its sister genus picea, high-density genetic and qtl maps have been prepared using tgop in loblolly and maritime pine. *minor linkage groups genome length and map coverage the black spruce genome length estimated in our study is cm. this is comparable with the genome length of or cm reported for black × red spruce hybrids based on a composite map of f and/or bc mapping populations and that of cm estimated for black × red spruce controlled cross hybrids from a bc mapping population. these results are consistent with the length of the genetic map for black spruce that is likely to be between and cm. the length of the black spruce genetic map observed in our study is about 15% shorter than that reported for norway spruce and white spruce maps. the genome size of norway spruce and white spruce is % and %, respectively, higher than that of black spruce. although no direct relationships between the nuclear dna contents and genetic map lengths apparently exist, the shorter genetic map length in black spruce than in norway spruce or white spruce is apparently consistent with its comparatively smaller genome size. our results suggest that the genome length observed in our study covers more than 97% of the estimated black spruce genome length. thus, the black spruce genetic map reported here can very well be considered as almost complete. this is the highest map coverage so far for any picea species. the estimated length and the extent of coverage of genetic maps in different species could vary owing to differences in the mapping populations used, variation in recombination rates of the parents, and the number and types of markers used in linkage map construction. the mapping pedigree and markers used in our study are different from those used in norway spruce and white spruce. linkage groups and marker density in our study, the paternal, maternal, and consensus linkage maps consistently coalesced into linkage groups, corresponding to the haploid chromosome number in black spruce. by contrast, in all other studies reported on genome mapping in the genus picea, with one exception, maternal, paternal, and/or consensus map did not coalesce into linkage groups. the consensus map reported here has markers distributed over linkage groups, which represents an almost complete coverage of the black spruce genome, as the number of linkage groups corresponds to the haploid chromosome number. the average distance observed among adjacent markers mapped for the genetic map of black spruce in our study is comparable with or lower than that reported for the composite map of black spruce × red spruce complex, as well as lower than that reported for norway spruce. however, the marker density of the black spruce genetic map reported here is the highest for any genetic map based on a single cross in the genus picea, as well for the maternal and paternal genetic maps. marker systems the genetic map of black spruce was constructed using aflp, sampl, ssr, and estp marker systems. the aflp and sampl systems provided a sufficient number of anonymous polymorphic and informative markers to construct a high density to near-saturated genetic map, whereas the ssr and estp systems provided highly informative and co-dominant markers. although ssr and estp markers, due primarily to their codominant nature, would be preferred for genome mapping, availability of limited numbers of these markers precludes constructing a high density to saturated genetic map in conifers using only these markers. we mapped aflp markers that were resolved by primer combination, showing an average multiplex ratio of markers per primer combination. this multiplex ratio is comparable to that observed in norway spruce and loblolly pine. we mapped sampl markers on different linkage groups. the multiplex ratio for sampl markers was comparable to that observed for aflp markers. the only other report where sampl markers have been used for genetic linkage mapping in conifers is for norway spruce, where sampl markers were mapped using two primer combinations. a large number of aflp and sampl markers segregated in the 3: ratio, which suggests that the parental genomes are highly heterozygous. as aflp and sampl markers were dominant, the 3: segregating markers were useful for aligning the parental maps to construct the consensus map, which cannot be established directly. also the intercross markers can help to identify additional linkage groups that were not represented in the parental maps. ssr loci provided highly informative markers. forty-two ssr loci were mapped onto linkage groups; of these were highly informative for integrating the maternal and paternal maps to construct the consensus map. the mapped cdna-based ssr markers are excellent candidates for comparative and composite mapping because these markers are expected to show high intraspecific homology and high interspecific orthology. also these markers allowed mapping of known genes on seven different linkage groups. most est primer pairs resolved multilocus patterns, which is not surprising given the occurrence of multigene families in conifers. also, the rate of polymorphism observed in estps was very low, which suggests that more powerful methods, such as detection of single nucleotide polymorphisms can be used to increase the resolution of polymorphism. nevertheless, the mapped estp loci along with cdna-based ssr markers provide good candidates for comparative mapping in picea, pinaceae, or other conifers. the genetic map presented here is the first-generation genetic map for black spruce, which provides a framework to map snp and other markers in the second-generation genetic map. we are planning to map snp markers from candidate genes and genes expressed differentially in response to climate change and snps mapped by pavy et al. onto this map in future. this is further discussed below under the future perspective section. the mapped snp markers from expressed genes are quite useful for qtl and association mapping of relevant traits because allelic variation in the genes could be linked or associated with trait phenotypes. however, these markers represent a very small proportion of the spruce genome. assuming expressed genes of average kb size and genome size of gbp in black spruce, the transcribed genome represents less than % of the whole genome. snps in about candidate genes are normally mapped, further reducing the proportion of the genome sampled to less than %, although the mapped genes may have genome-wide distribution. the anonymous markers, such as aflp and sampl markers generally provide a whole-genome scan; thus a genetic map using these markers may cover a large proportion of the genome, if not the whole genome. however, anonymous markers such as aflp and sampl, have limitations in tagging genes controlling traits of interest via qtl and association mapping because variation in these markers may not represent functional genetic variation. nevertheless, the black spruce genetic map reported here provides a valuable genomic resource in picea and pinaceae. clustering of markers even though only those markers segregating in mendelian ratios and not those showing a distorted segregation were used for the linkage analysis, clustering of aflp and sampl markers was detected in the linkage groups of black spruce. these results agree with the clustering of aflp markers reported for genetic maps of picea abies, pinus taeda, and pinus sylvestrisl, but in contrast to random distribution of aflp markers in the genetic maps reported for norway spruce and black × red spruce hybrids. it should be noted that the study of scotti et al. was specifically performed to examine the distribution of marker classes in a genetic linkage map of norway spruce. the non-random distribution of markers may be caused by non-random and unequal crossing over and recombination along the chromosome length. the recombination is suppressed in the centromeric and heterochromatic pericentromeric regions, and the presence of heterochromatin in pericentromeric regions is a general feature of plant chromosomes. assuming a random distribution of markers, low levels of meiotic recombination may well cause markers that are physically well separated, to cluster on a linkage map. future perspectives the genetic map reported here is suitable for constructing a composite map of two tgop in black spruce and for the envisaged comparative mapping with red spruce and black spruce × red spruce hybrid. the mapped est-based microsatellites and estps will provide very useful markers for this work. we are also mapping additional est-based ssr markers. black spruce and red spruce are highly genetically related species but their evolutionary relationships are controversial. comparative genome mapping may shed some light on comparative genome organization and evolution of these species. the map could also be used for comparative mapping with other genera of pinaceae. we are genotyping and phenotyping the larger mapping populations of the tgop used in this study and another tgop of black spruce for qtl mapping of traits related to growth and adaptation to climate change. we plan to use about snps from candidate genes and genes expressed differentially in response to climate change conditions as well as snps mapped by pavy et al. for our qtl mapping work, using a high throughput snp genotyping platform such as illumina's golden gate genotyping assay http://www.illumina.com/technology/goldengate_genotyping_assay.ilmn. thus, the technologies, markers and genetic map developed in the present study provide an invaluable genomic resource for basic and applied genomics studies.  we have constructed the first high density to near-saturated genetic linkage map of black spruce, with above 97% genome coverage. the maternal, paternal, and consensus maps in our study consistently coalesced into linkage groups, corresponding to the haploid chromosome number of in the genus picea. also, this is the first genetic map based on a three-generation outbred pedigree in the genus picea. the genome length in p. mariana is likely to be about cm. the genetic maps developed in our study can serve as a reference map for various genomics studies and applications in picea and pinaceae. it will provide a foundation and a valuable resource for comparative mapping, constructing composite maps, and mapping quantitative trait loci and determining the genetic basis of complex quantitative traits of interest, such as growth and adaptation to climate change. 
the genetic maps developed in our study can serve as a reference map for various genomics studies and applications in picea and pinaceae. our goal was to construct a high-density genetic linkage map of black spruce, which is a predominant, transcontinental species of the north american boreal and temperate forests, with high ecological and economic importance. we have developed a near-saturated and complete genetic linkage map of black spruce using a three-generation outbred pedigree and amplified fragment length polymorphism, selectively amplified microsatellite polymorphic loci, expressed sequence tag polymorphism, and microsatellite markers. maternal, paternal, and consensus genetic linkage maps were constructed. we have constructed the first high density to near-saturated genetic linkage map of black spruce, with greater than 97% genome coverage. genetic maps provide an important genomic resource for understanding genome organization and evolution, comparative genomics, mapping genes and quantitative trait loci, and associating genomic segments with phenotypic traits. total estimated length of the genetic map was cm, with an average of one marker every cm. the maternal, paternal and consensus genetic maps aligned almost perfectly. also, this is the first genetic map based on a three-generation outbred pedigree in the genus picea. spruce genomics work is quite challenging, mainly because of extremely large size and highly repetitive nature of its genome, unsequenced and poorly understood genome, and the general lack of advanced-generation pedigrees. the maternal, paternal, and consensus maps in our study consistently coalesced into linkage groups, corresponding to the haploid chromosome number of in the genus picea. the maternal map had and the paternal map markers distributed over linkage groups each. the genome length in p. mariana is likely to be about cm. the consensus map consisted of markers distributed over linkage groups, and covered almost the entire black spruce genome.
 genetic maps provide an important genomic resource for understanding genome organization and evolution, comparative genomics, mapping genes and quantitative trait loci, and associating genomic segments with phenotypic traits. spruce genomics work is quite challenging, mainly because of extremely large size and highly repetitive nature of its genome, unsequenced and poorly understood genome, and the general lack of advanced-generation pedigrees. our goal was to construct a high-density genetic linkage map of black spruce, which is a predominant, transcontinental species of the north american boreal and temperate forests, with high ecological and economic importance.  we have developed a near-saturated and complete genetic linkage map of black spruce using a three-generation outbred pedigree and amplified fragment length polymorphism, selectively amplified microsatellite polymorphic loci, expressed sequence tag polymorphism, and microsatellite markers. maternal, paternal, and consensus genetic linkage maps were constructed. the maternal, paternal, and consensus maps in our study consistently coalesced into linkage groups, corresponding to the haploid chromosome number of in the genus picea. the maternal map had and the paternal map markers distributed over linkage groups each. the consensus map consisted of markers distributed over linkage groups, and covered almost the entire black spruce genome. the mapped markers included aflps, sampl, microsatellites, and estps. total estimated length of the genetic map was cm, with an average of one marker every cm. the maternal, paternal and consensus genetic maps aligned almost perfectly.  we have constructed the first high density to near-saturated genetic linkage map of black spruce, with greater than 97% genome coverage. also, this is the first genetic map based on a three-generation outbred pedigree in the genus picea. the genome length in p. mariana is likely to be about cm. the genetic maps developed in our study can serve as a reference map for various genomics studies and applications in picea and pinaceae. 