21Massively parallel sequencing has revolutionized the field of genetics by providing 22 comparatively high-resolution insights into whole genomes for large number of species so far. 23 However, whole-genome resequencing of many conspecific individuals remains cost-prohibitive for 24 most species. This is especially true for species with very large genomes with extensive genomic 25 redundancy, such as the genomes of coniferous trees. The genome assembly for the conifer Norway 26 spruce (Picea abies) was the first published draft genome assembly for any gymnosperm. Our goal was 27 to develop a dense set of genome-wide SNP markers for Norway spruce to be used for assembly 28 improvement and population studies. From 80,000 initial probe candidates, we developed two 29 partially-overlapping sets of sequence capture probes: one developed against 56 haploid 30 megagametophytes, to aid assembly improvement; and the other developed against 6 diploid needle 31 samples, to aid population studies. We focused probe development within genes, as delineated via the 32 annotation of ~67,000 gene models accompanying P. abies assembly version 1.0. The 31,277 probes 33 developed against megagametophytes covered 19,268 gene models (mean 1.62 probes/model). The 34 40,018 probes developed against diploid tissue covered 26,219 gene modules (mean 1.53 35 probes/model). Analysis of read coverage and variant quality around probe sites showed that initial 36 alignment of captured reads should be done against the whole genome sequence, rather than a subset of 37 probe-containing scaffolds, to overcome occasional capture of sequences outside of designed regions. 38 All three probe sets, anchored to the P. abies 1.0 genome assembly and annotation, are available for 39 download. 40 41 61 Depending on the hybridization technology, varying numbers of probes can be used. For humans, 62 multiple technologies are available which contain probes sufficient to capture whole exomes (Clark et 63 al., 2011; Shigemizu et al., 2015). Such comprehensive approaches can be used successfully in model 64 species with well-annotated genomes (Fu et al., 2013; Zhou et al. 2012; Zhou et al. 2014). However, 65 because sequence capture relies largely on the accuracy of genome annotations and the uniqueness of 66 probe targets, it may exhibit reduced efficiency when applied to non-model species with incomplete 67 annotations and/or species with complex genomes containing much repetitive content (Neves et al., 123 comprise the final probe set. 124 Plant material and DNA extraction. 125 Haploid genomic DNA was extracted from 52 megagametophytes. The megagametophytes 126 were excised from open pollinated seeds of Z4006 ramets (Z4006: the Norway spruce reference 127 sequence individual), under the microscope in order to avoid diploid tissue. DNA was extracted with 128 the NucleoSpin® Plant II kit, (Macherey-Nagel, http://www.mn-net.com). After several modifications 129 of the manufacture's recommended protocol, we achieved the highest concentration of DNA from 130 megagam...

Study of natural variation is an efficient method to elucidate how plants adapt to local climatic conditions, a key process for the evolution of a species. However, it is challenging to determine the genetic basis of adaptive variation especially in forest trees which have large and complex genomes. Norway spruce is a shade tolerant conifer in which the requirement of far-red light for growth increases latitudinally northwards. In the current work, hypocotyl-length followed a latitudinal cline in response to SHADE (low red:far-red ratio). RNA-sequencing revealed differential gene expression in response to SHADE, between a southern and a northern natural population in Sweden. Exome capture included analysis of uniquely large data set (1654 trees) that revealed missense variations in coding regions of nine differentially expressed candidate genes, which followed a latitudinal cline in allele and genotype frequencies. These genes included five transcription factors involved in vital processes like bud-set/bud-flush, lignin pathway and cold acclimation, and other genes that take part in cell-wall remodeling, secondary cell-wall thickening, response to starvation and immunity. Findings from this work primarily suggests that the northern populations of Norway spruce are better adapted towards disease resistance under shade by up-regulation of lignin pathway that is linked to immunity and it forms concrete basis for local adaptation to light quality in Norway spruce, one of the most economically important conifer tree species in Sweden.