# Source paper archive - Source title: Genome-wide latitudinal variation associated with defence and reproductive pathways linked to Norway spruce adaptation - Author(s): Ranade, S. S., Garcia-Gil, M. R. - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.11.06.764856v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.11.06.764856v1.full.pdf - PDF SHA-256: 3067d57ad68c1bf156d8666795bde66164b235da8b8acefce68eb9279c1ab693 - Converter: pymupdf4llm 0.0.27 - Limits: Automated PDF text extraction; reading order, equations, tables and formatting may be incomplete or incorrect. Images are omitted, and no OCR was performed. Markup and links are neutralized for safe downloading. Consult the original PDF for the authoritative version. --- [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 **Genome-wide latitudinal variation associated with defence and reproductive pathways** 2 **linked to Norway spruce adaptation** 3 4 Sonali Sachin Ranade, María Rosario García-Gil * 5 Umeå Plant Science Centre (UPSC), Department of Forest Genetics and Plant Physiology, 6 Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden 7 8 - Corresponding author 9 [Sonali Sachin Ranade: Sonali.Ranade@slu.se](mailto:Sonali.Ranade@slu.se) 10 [María Rosario García-Gil: M.Rosario.Garcia@slu.se](mailto:M.Rosario.Garcia@slu.se) 11 12 **Highlights** 13 Genome-wide analysis from exome capture revealed natural variation in genes associated with 14 defence and reproductive pathways in Norway spruce population across Sweden. Variation in 15 allele frequencies of SNPs that follows a latitudinal cline, despite of weak population structure 16 with low genetic differentiation, may represent cues for local adaptation. Knowledge gained 17 can be applied to design future sustainable breeding strategies in forestry. 18 19 **Abstract** 20 Environmental cues modulate the natural variations in genes that outline local adaptation in 21 plants for optimal survival. Genome-wide analysis using exome capture dataset in Norway 22 spruce ( _Picea abies_ L. Kast) in Sweden revealed natural allelic variation in coding regions of 23 genes associated with defence and reproductive pathways that follow a latitudinal cline, despite 24 of the weak population structure with a low genetic differentiation. Steepest clines in the allele 25 frequencies of missense SNPs from coding region (cSNPs) were detected in _60S RIBOSOMAL_ 26 _PROTEIN L18_ ( _RPL18C_ ), a gene involved in plant survival and defence/stress responses and 27 _LONG AFTER FAR-RED 3_ ( _LAF3_ ) that mediates light responses. This was closely followed 28 by cline in _ZINC 4 FINGER DNA 3'-PHOSPHOESTERASE_ ( _ZDP_ ), involved in DNA repair 29 and DNA demethylation pathways. Flowering genes that displayed latitudinal cline in cSNPs 30 included _BARELY ANY MERISTEM 1_ ( _BAM1_ ), _REDUCED VERNALIZATION RESPONSE 1_ 31 ( _VRN1_ ), _FLOWERING CONTROL LOCUS A_ ( _FCA_ ) and _TERMINAL FLOWER 1_ ( _TFL1_ ) 32 among few others. These variations may contribute to differential functioning of the 33 defence/flowering genes, enabling the tree to thrive at its best under given local environmental 34 conditions. The clines provide evidence towards local adaptation in these Norway spruce 1 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 35 populations across Sweden; it coincides with the latitudinal variation in climatic conditions 36 including temperature, day length and light quality, along with existing cline e.g. differential 37 growth and defence response, and bud-burst/bud-set. The knowledge can be applied in forest 38 industry to design future sustainable breeding strategies e.g. assisted migration, in the context 39 of inevitable climate change. 40 41 **Keywords** 42 **Cline, cSNP, defence, flowering, local adaptation, natural variation, Norway spruce** 2 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 43 **Introduction** 44 A latitudinal cline is defined as a measurable gradient of a single trait or character in a particular 45 species, across its geographical range which is shaped by natural selection matching the 46 existing climatic conditions. Clines provide information on patterns of genetic adaptation to 47 varying environmental factors e.g. temperature, photoperiod and light quality. Local adaptation 48 occurs when resident populations evolve divergent phenotypic traits allowing the organism to 49 thrive optimally under the prevailing environmental conditions including biotic and abiotic 50 factors. Genome-wide screening for latitudinal clines is an efficient method that can be applied 51 to identify novel genetic changes which could be correlated with shifts in geography and 52 climate, thereby facilitating understanding of local adaptation in the species. For example, 53 identification of new locus contributing to phenotypic differences was discovered recently in 54 snapdragons by whole-genome cline scans in hybrid zones (Field et al., 2026). 55 56 FR-enriched light is perceived as shade by the trees, e.g. under canopy, most of the R light is 57 absorbed by the plant pigments while FR is reflected leading to reduction in the R:FR ratio 58 (Ballare et al., 1987). A low R:FR ratio triggers the plant's shade avoidance response – the 59 survival mechanism by altering the growth patterns (Ranade et al., 2022, Ranade et al., 2019). 60 Light quality varies with latitude across Sweden; the ratio of red (R) to far-red (FR) light (R:FR) 61 decreases as you move from south to north, leading to an increased amount of FR (twilight) in 62 the northern latitudes which is similar to shade-like condition (Nilsen, 1985). This gradient 63 shapes how native flora including local Norway spruce populations regulate seasonal growth 64 and flowering during the extended daylight periods comprising longer exposure to FR light in 65 the northern latitudes during the growing season. Norway spruce exhibits a cline for growth in 66 response to light quality in the Swedish populations (Clapham et al., 1998, Molmann et al., 67 2006, Ranade and García-Gil, 2021). 68 69 Norway spruce is a shade tolerant conifer species, meaning it can establish and compete well 70 under shaded conditions (Ranade and García-Gil, 2023, Grebner et al., 2021, Ranade et al., 71 2022, Ranade et al., 2019). The species shows a requirement for FR light to maintain growth 72 that follows a latitudinal gradient (Clapham et al., 1998, Molmann et al., 2006, Ranade and 73 García-Gil, 2021). Norway spruce has evolved distinct genetic variations that follow a 74 latitudinal cline in several genes e.g. _phytochrome_, _cryptochrome_, _expansin B3_ and _MYB_ 75 _domain protein 3_ that reflects local adaptation in the species (Ranade and García-Gil, 2023, 76 Ranade and García-Gil, 2021). For example, cSNP variations in phytochromes which are the 3 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 77 central regulators of the light signalling pathway, interpret regional differences in far-red 78 signals, thus adapting to the local light conditions across Sweden. Lignin synthesis and growth 79 are influenced by light quality; Norway spruce displays latitudinal variation in cSNPs in the 80 genes from lignin biosynthesis pathway and variation in their expression along with differential 81 lignin formation, influencing disease resilience (Ranade et al., 2022). Northern populations of 82 Norway spruce are slow growing but disease resilient as compared to the southern populations; 83 the northern trees have enhanced lignin synthesis and expression of higher number of defence 84 related genes during growth seasons in response to the prevailing light conditions namely 85 higher exposure periods to FR light (Ranade et al., 2022). Phenological traits like bud-burst 86 and bud-set driven by environmental cues like temperature also show a strong latitudinal cline 87 for bud phenology in Norway spruce (Chen et al., 2014, Skroppa et al., 2010, Mikola, 1982). 88 The northern populations of Norway spruce have adapted to shorter growing seasons by 89 initiating bud-set earlier and requiring less accumulated heat (lower temperature) to trigger 90 spring bud-burst than southern populations. 91 92 Understanding clines, particularly involving local adaptation is challenging yet significant for 93 managing long-lived species like Norway spruce that has widespread distributions and is 94 economically important species for the forest industry in Sweden. Related information in this 95 domain could be used in tree breeding methods to design assisted migration strategies as trees 96 are genetically programmed for specific growing season length, frost tolerance and light 97 quality. This can facilitate selection of locally adapted genotypes that optimise the balance 98 between high survival rates matching the changing climate and sustained productivity, for 99 breeding programs. In addition, it can also facilitate selection of families resistant to region 100 specific diseases or pathogen resilient varieties in general. 101 102 The current investigation aims at genome-wide identification of clinal variation in genes in 103 Norway spruce populations across Sweden with the exome capture dataset, to explore the 104 genetic and molecular basis of clinal adaptations. The study includes exome capture data from 105 1654 unrelated Norway spruce trees sampled across a wide latitudinal range in Sweden (55 106 67°N) that has variation in the photoperiod and temperature during the growth season. This 107 data is most ideal to examine the SNP variation in a series of genes that may be correlated to 108 the variation in the environmental/climatic parameters. 109 4 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 110 **Material and Methods** 111 _Plant material_ 112 1654 individuals from natural forests (with unrelated parents), from different latitudes across 113 Sweden were recruited in this study (Ranade and García-Gil, 2023). Individuals were divided 114 into six populations considering the same principle described in Ranade and García-Gil, 2023 115 that did not follow any specific strategy, however it ensured that all the groups were continuous 116 populations allowing free pollen exchange (Ranade and García-Gil, 2023). Six populations 117 were represented by groups S1-S6. S1 comprised 245 trees from latitudes 55-57°N, S2 – 213 118 trees from latitude 58°N, S3 – 187 trees from latitudes 59-60°N, S4 – 213 trees from latitudes 119 61-62°N, S5 – 573 trees from latitudes 63-64°N and S6 – 223 trees from latitudes 65-67°N. 120 These individuals represent a subset of a study that reports the structure of Swedish populations 121 of Norway Spruce, showing two distinct groups – one includes central and southern 122 populations in Sweden, while the other includes the populations from northern Sweden (Chen 123 et al., 2021). 124 125 _SNP detection, diversity and detection of latitudinal cline_ 126 Norway spruce genome (v1.0) genome retrieved from the PlantGenIE resource (Sundell et al., 127 2015) was used as the reference genome for this analysis. SNP variation was derived by using 128 exome capture, described in Baison et al. (Baison et al., 2019). Only bi‐allelic missense cSNPs 129 i.e. missense SNP from coding regions, were considered for analysis in this study. Missense 130 cSNP represents a change in the protein-coding gene resulting in the substitution of one amino 131 acid to another. This alteration in the amino acid leads to a change in the sequence of the 132 encoded protein, which can impact its primary structure, protein folding, and stability, 133 potentially leading to modification in its mode of function. The change in the amino acid 134 possibly has a greater effect if it involves a change from hydrophilic to hydrophobic amino acid 135 or polar to non-polar and vice-versa. Filtering criteria applied to VCF files was as follows, ‐‐ 136 min‐alleles 2 ‐‐max‐alleles 2 ‐‐maf 0.01 ‐‐remove‐indels ‐‐minQ 10 ‐‐max‐missing 0.9. 137 138 Allele and genotype frequencies across the six populations were determined using SNPassoc 139 (Gonzalez et al., 2007) and analysis of variance and Tukey's posthoc tests (Bonferroni _p values_ ) 140 were applied to determine the statistical significance of their differences. When there was a 141 latitude-wise consecutive increase/decrease in allele frequency across the six populations, the 142 allele was documented to exhibit a cline. The steepness of cline in the given allele was 5 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 143 calculated as the difference in allele frequency between the southern-most and the northern 144 most populations, e.g. 0.6 was the steepest cline detected in this study, while 0.1 was considered 145 to be the milder one. Latitudinal cline was categorised as standard cline and trend cline. In a 146 standard cline, the allele frequencies either increased or decreased in a steady manner with each 147 population from south to north, e.g. in case of the cSNPs with steepest clines (Figure 1 and 148 Figure 2). The trend cline either shows an increasing trend or decreasing trend in the allele 149 frequencies in the populations from south to north, meaning the increase or decrease in the 150 allele frequencies does not occur serially or latitude wise, instead it shows a trendline. For 151 example, a cSNP (Thr125Ala, Polar hydrophilic > Non-polar hydrophobic) from LHCB7 152 shows high cline steepness of 0.4; however, although populations 1 to 5 show a steady increase 153 in the allele frequency, there is a decrease in allele frequency in population 6 as compared to 154 the population 5 (Supplementary file1 – Table S1; Supplementary file2 – Figure S21). Genetic 155 diversity among the six Norway spruce populations across Sweden, was determined by Weir 156 and Cockerham F ST estimates. This included 238 512 biallelic missense SNPs from 1654 157 Norway spruce trees. 158 159 _Gene ontology (GO) enrichment analysis_ 160 Gene ontology (GO) enrichment analysis was performed by using ShinyGO 0.85.1, with a p 161 value cut-off of 0.05 (FDR), using the _Arabidopsis_ database, as _Arabidopsis_ is the most well 162 studied plant model (Ranade et al., 2022). GO:0003700 (DNA-binding transcription factor 163 activity) was referred for deriving the transcription factors and they were grouped into types 164 using PlantTFDB (Jin et al., 2017). 6 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 165 **Results** 166 The filtering of the raw VCF file retained 238 512 biallelic SNPs. 37 912 missense cSNPs were 167 annotated (PlantGenIE resource (Sundell et al., 2015)) having a representative _Arabidopsis_ 168 gene, representing 10161 MA ids (generally, one MA id represents one gene). Finally, 2935 169 missense cSNPs from the 1654 individuals across six populations, showed standard latitudinal 170 cline in their allele/genotype frequencies. These SNPs belonged to 2084 annotated genes from 171 Norway spruce including flowering and defence related genes. Genes including missense 172 cSNPs with the steepest standard clines are included in Table 1 and Table 2, while a complete 173 list of all the missense cSNPs showing standard and trend cline is represented in the 174 Supplementary file1 – Table S1. Alignments of the Norway spruce genes with the model plant 175 _Arabidopsis_ for the genes that show steepest clines are included in Supplementary file2 (Figure 176 S1-S20). The alignments file includes the details of the variation that shows standard cline e.g. 177 nucleotide/codon/amino acid alteration and the cline steepness. 178 179 Cline steepness in the allele frequencies of an allele was calculated as the difference in allele 180 frequency between the southern-most and the northern-most populations. Steepest standard 181 clines in the allele frequencies of missense cSNPs were detected in _60S RIBOSOMAL_ 182 _PROTEIN L18_ ( _RPL18C_ ), a gene involved in plant survival and defence/stress responses 183 (Figure 1) and _LONG AFTER FAR-RED 3_ ( _LAF3_ ) that mediates light responses (Figure 2). 184 This was closely followed by cline in _ZINC 4 FINGER DNA 3'-PHOSPHOESTERASE_ ( _ZDP_ ), 185 a gene involved in DNA repair and DNA demethylation pathways (Table 1), which showed 186 cline in three cSNPs. cSNPs from genes involved in ATP production ( _COENZYME Q9_ ; _COQ9_ ), 187 regulation of abiotic stress ( _SERINE/ARGININE RICH-LIKE PROTEIN 45A_ ; _SR45A_ ), 188 oxidative stress ( _OXIDATION RESISTANCE 1_ ; _OXR1_ ), osmotic balance ( _ALUMINUM-_ 189 _ACTIVATED MALATE TRANSPORTER 9_ ; _ALMT9_ ), pollen development ( _ATP-BINDING_ 190 _CASSETTE G26_ ; _ABCG26_ ) and defence response ( _TERPENE SYNTHASE 02_ ; _TPS02_ ) also 191 exhibited steep clines (Table 1). The allele frequencies, genotype frequencies and the p-values 192 for Hardy-Weinberg Equilibrium (HWE) from the missense cSNPs with steepest clines are 193 included in Supplementary file1 – Table S2. 194 195 Genes from reproductive pathways involved in flowering that showed the steepest clines in 196 allele frequencies of missense cSNPs included _BARELY ANY MERISTEM 1_ ( _BAM1_ ), 197 _REDUCED VERNALIZATION RESPONSE 1_ ( _VRN1_ ), closely followed by _JMJC DOMAIN-_ 198 _CONTAINING PROTEIN 27_ ( _JMJ27_ ) and _LATE MERISTEM IDENTITY2_ ( _LMI2_ ) (Table 2). 7 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 199 _FLOWERING CONTROL LOCUS A_ ( _FCA_ ) and _TERMINAL FLOWER 1_ ( _TFL1_ ) involved in 200 transition from vegetative growth to flowering and regulation of flowering time, also displayed 201 significant clines (Table 2). The allele frequencies, genotype frequencies and the p-values for 202 HWE from the missense cSNPs from flowering genes are included in Supplementary file1 – 203 Table S3. Approximately 50% of cSNPs with steep clines (Table 1, Table 2) bear a change in 204 the amino acid from hydrophilic to hydrophobic amino acid or polar to non-polar and vice 205 versa, indicating modification in the mode of function of the encoded protein. 206 207 The available version of Norway spruce genome (v1.0) during the analysis (Sundell et al., 208 2015), was fragmented, yet the sequence alignments (Supplementary file2 – Figure S1-S20) 209 and tools applied on the PlantGenIE resource (Sundell et al., 2015) to identity the Norway 210 spruce gene, represent consistent results. Due to fragmentation, the gene sequences are not 211 complete, and some genes are proposed to be contained across two separate contigs. For 212 example, the genes _RPL18C_, _LAF3_ and _COQ9_ are contained across two separate contigs 213 (Supplementary file2 – Figure S1-S4, Figure S6-S7). Though, these could also potentially be 214 the homologues of RPL18C, LAF3 and _COQ9_ owing to fragmented genome. In any case, these 215 gene fragments contain variations that show cline. Few of the genes show cline in multiple 216 cSNPs; some cSNPs display steep cline while others represent milder clines. _LAF3_ shows two 217 missense variants with a cline steepness of 0.6 and 0.4, while and _COQ9_ shows one missense 218 variant with cline steepness of 0.5 and four other variants with cline steepness of 0.1 (Table1, 219 Supplementary file2 – Figure S3-S4, Figure S6-S7). _SR45a_, _OXR1_ and _TPS02_ contain missense 220 variants with cline steepness of 0.1, 0.4 and 0.3, respectively (Table1, Supplementary file1 – 221 Table S1; Supplementary file2 – Figure S8, Figure S10, Figure S12). Likewise, with reference 222 to flowering genes (Table 2) _FCA_ has four additional missense variants, each with a cline 223 steepness of 0.1 while homologues of _LMI2_ (Supplementary file2 – Figure S16-S18), 224 _AGAMOUS-LIKE 20_ ( _AGL20_ ) and _Apetala 2_ ( _AP2_ ) have variants representing mild (0.1) to 225 very mild cline (<0.1) (Supplementary file1 – Table S1). 226 227 Gene ontology (GO) enrichment analysis was conducted with 2084 genes that were annotated 228 in Norway spruce, harbouring 2935 missense cSNPs from 1654 individuals across the six 229 populations included in this study. GO analysis for Biological Process indicated higher fold 230 enrichment for developmental processes involved in reproduction, along with response to 231 hormone, endogenous stimulus and processes related to post-embryonic development, which 232 can be correlated to the clines detected in the flowering genes (Supplementary file2 – Figure 8 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 233 S22). GO analysis for Molecular Function showed significantly higher representation of genes 234 involved in nucleic acid binding indicating active transcription and DNA repair mechanisms 235 (Supplementary file2 – Figure S23). This coincides with one of the steepest clines detected in 236 _ZDP_, a gene involved in DNA repair. Cellular Compartment GO analysis included higher 237 representation of genes localised in the plastids, while higher fold enrichment was represented 238 by nuclear body category (Supplementary file2 – Figure S24). 239 240 MYB family members represented the highest numbers among all the transcription factors that 241 followed a cline in the Norway spruce populations included in this study (Figure 3). MYBs are 242 primarily involved in secondary cell wall formation, lignin deposition, wood development and 243 stress response (Wu et al., 2024, Ranade and García-Gil, 2024). MYBs were closely followed 244 by EFRs and bHLH family members. ERFs act downstream of the ethylene signalling pathway 245 in addition to response to stress and defence (Wu et al., 2022) while bHLHs are involved in 246 light signalling, regulation of growth and development (Zuo et al., 2023). 247 248 Weir and Cockerham F ST estimates for six populations of Norway spruce across Sweden 249 showed low values (Table 3). 250 251 Figure 1 252 253 **Figure 1** Allele and genotype frequencies of SNP variation in RPL18C gene showing cline 254 across Norway spruce populations in Sweden. (a) Allele frequencies of Leu74Phe. (b) 255 Genotype frequencies of Leu74Phe. One-way ANOVA and Tukey’s post-hoc test with the 256 genotype frequencies showed significant differences across populations; Tukey’s post-hoc 257 categorization is indicated above the bars. 9 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 258 259 **Figure 2** Allele and genotype frequencies of SNP variation in LAF3 gene showing cline across 260 Norway spruce populations in Sweden. (a) Allele frequencies of Leu35Ser. (b) Genotype 261 frequencies of Leu35Ser. One-way ANOVA and Tukey’s post-hoc test with the genotype 262 frequencies showed significant differences across populations; Tukey’s post-hoc categorization 263 is indicated above the bars. 264 265 266 **Figure 3** Number of transcription factors of each type, with missense cSNPs showing clinal 267 variation across Norway spruce populations in Sweden. 10 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 268 **Table 1** Genes showing steep latitudinal cline in cSNPs in Norway spruce populations across Sweden 269 primarily belong to defence/DNA repair pathways. Change in the amino acid property e.g. polar to non 270 polar/ hydrophilic to hydrophobic and vice-versa is indicated in bold. |Genename|SprucegeneID<br>(PlantGenIE)/Arabidopsis<br>geneID(TAIR)|Variation/Cline<br>steepness|Function|Reference| |---|---|---|---|---| |_60S RIBOSOMAL_<br>_PROTEIN L18_<br>(_RPL18C_)|MA_3674g0020/<br>AT5G27850|CTC > TTC, Leu74Phe<br>Non-polar hydrophobic ><br>Non-polar hydrophobic<br>Cline steepness: 0.6|Component of 60S<br>subunit, facilitates<br>ribosome assembly and<br>translation, plant survival<br>and defence/stress<br>response.|(Tao et al., 2024)| |_LONG AFTER FAR-_<br>_RED 3_ (_LAF3_)|MA_9446113g0010/<br>AT3G55850|TTA > TCA, Leu35Ser<br>**Non-polar hydrophobic**<br>**> Polar hydrophilic**<br>Cline steepness: 0.6|Mediates Phytochrome A<br>dependent responses<br>under far-red light e.g.<br>hypocotyl growth<br>inhibition.|(Hare et al.,<br>2003)| |_ZINC 4 FINGER DNA_<br>_3'-_<br>_PHOSPHOESTERASE_<br>(_ZDP_)|MA_39122g0010/<br>AT3G14890|CCA > CGA, Pro22Arg<br>**Non-polar hydrophobic**<br>**> Polar hydrophilic**<br>Cline steepness: 0.5|Involved in DNA repair<br>and DNA demethylation<br>pathways, maintenance of<br>epigenome and genome<br>stability.|(Li et al., 2025)| |_ZINC 4 FINGER DNA_<br>_3'-_<br>_PHOSPHOESTERASE_<br>(_ZDP_)|MA_39122g0010/<br>AT3G14890|AAC > GAC, Asn31Asp<br>Polar hydrophilic > Polar<br>hydrophilic<br>Cline steepness: 0.5|AAC > GAC, Asn31Asp<br>Polar hydrophilic > Polar<br>hydrophilic<br>Cline steepness: 0.5|AAC > GAC, Asn31Asp<br>Polar hydrophilic > Polar<br>hydrophilic<br>Cline steepness: 0.5| |_ZINC 4 FINGER DNA_<br>_3'-_<br>_PHOSPHOESTERASE_<br>(_ZDP_)|MA_39122g0010/<br>AT3G14890|TGT > TAT, Cys69Tyr<br>Polar hydrophilic > Polar<br>hydrophilic<br>Cline steepness: 0.5|TGT > TAT, Cys69Tyr<br>Polar hydrophilic > Polar<br>hydrophilic<br>Cline steepness: 0.5|TGT > TAT, Cys69Tyr<br>Polar hydrophilic > Polar<br>hydrophilic<br>Cline steepness: 0.5| |_COENZYME Q9_<br>(_COQ9_)|MA_9293920g0010/<br>AT1G19140|TCT > GCT, Ser77Ala<br>**Polar hydrophilic >**<br>**Non-polar hydrophobic**<br>Cline steepness: 0.5|Involved electron transfer<br>respiratory chain crucial<br>for ATP production, in<br>mitochondria. Acts as<br>antioxidant.|(Xu et al., 2025)| |_SERINE/ARGININE_<br>_RICH-LIKE PROTEIN_<br>_45A_ (_SR45A_)|MA_42236g0030/<br>AT1G07350|AGC > ATC, Ser209Ile<br>**Polar hydrophilic >**<br>**Non-polar hydrophobic**<br>Cline steepness: 0.5|Abiotic stress - regulator<br>of tolerance to heat,<br>salinity and drought.|(Muthusamy et<br>al., 2020)| |_ATP-BINDING_<br>_CASSETTE G26_<br>(_ABCG26_)|MA_16472g0020/<br>AT3G13220|GAA > GAT, Glu34Asp<br>Polar hydrophilic > Polar<br>hydrophilic<br>Cline steepness: 0.5|Essential for pollen<br>development and<br>male fertility.|(Zhao et al.,<br>2016)| |_OXIDATION_<br>_RESISTANCE 1_ (_OXR1_)|MA_697287g0010/<br>AT4G39870|TCA > GCA, Ser129Ala<br>**Polar hydrophilic >**<br>**Non-polar hydrophobic**<br>Cline steepness: 0.5|Offers protection against<br>oxidative stress and<br>maintains genome<br>stability.|(Colombatti et<br>al., 2019)| |_ALUMINUM-_<br>_ACTIVATED MALATE_<br>_TRANSPORTER 9_<br>(_ALMT9_)|MA_10243484g0010/<br>AT3G18440|GTA > TTA, Val122Leu<br>Non-polar hydrophobic ><br>Non-polar hydrophobic<br>Cline steepness: 0.5|Regulation of stomatal<br>opening and osmotic<br>balance.|(De Angeli et al.,<br>2013)| |_TERPENE SYNTHASE_<br>_02_ (_TPS02_)|MA_10426551g0010/<br>AT4G16730|TGG > TTG, Trp43Leu<br>Non-polar hydrophobic ><br>Non-polar hydrophobic<br>Cline steepness: 0.5|Defence<br>against pathogens, insects,<br>herbivores.|(Huang et al.,<br>2010)| 11 271 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 272 **Table 2** Genes primarily involved in flowering showing steepest latitudinal cline in cSNPs in Norway 273 spruce populations across Sweden. Change in the amino acid property e.g. polar to non-polar/ 274 hydrophilic to hydrophobic and vice-versa is indicated in bold. |Genename<br>*TF(Transcription<br>Factor)|SprucegeneID<br>(PlantGenIE)/Arabidopsis<br>geneID(TAIR)|Variation/Clinesteepness|Function|Reference| |---|---|---|---|---| |_BARELY ANY_<br>_MERISTEM 1_<br>(_BAM1_)|MA_40935g0020/<br>AT5G65700|TAT > TCT, Tyr158Ser<br>Polar hydrophilic > Polar<br>hydrophilic<br>Cline steepness: 0.4|Required for floral<br>meristem development,<br>promotes anther<br>development.|(Hord et al., 2006,<br>DeYoung et al.,<br>2006)| |_BARELY ANY_<br>_MERISTEM 1_<br>(_BAM1_)|MA_40935g0020/<br>AT5G65700|GGC > GAC, Gly174Asp<br>**Non-polar hydrophobic >**<br>**Polar hydrophilic**<br>Cline steepness: 0.4|GGC > GAC, Gly174Asp<br>**Non-polar hydrophobic >**<br>**Polar hydrophilic**<br>Cline steepness: 0.4|GGC > GAC, Gly174Asp<br>**Non-polar hydrophobic >**<br>**Polar hydrophilic**<br>Cline steepness: 0.4| |_REDUCED_<br>_VERNALIZATION_<br>_RESPONSE 1_<br>(_VRN1_) <br>***TF type: B3**|MA_10430774g0010/<br>AT3G18990|TCA> TTA, Ser641Leu<br>**Polar hydrophilic > Non-**<br>**polar hydrophobic**<br>Cline steepness: 0.4|Floral activator,<br>involved intransition<br>from vegetative growth<br>to reproductive<br>development.|(King et al., 2013)| |_JMJC DOMAIN-_<br>_CONTAINING_<br>_PROTEIN 27_<br>(_JMJ27_)|MA_115863g0010/<br>AT4G00990|ACT > GCT, Thr1103Ala<br>**Polar hydrophilic > Non-**<br>**polar hydrophobic**<br>Cline steepness: 0.3|Repressor of flowering<br>and positive modulator<br>of immune responses|(Dutta et al., 2017)| |_LATE MERISTEM_<br>_IDENTITY2_ (_LMI2_) <br>*** TF type: R2R3**<br>**MYB**|MA_128244g0010/<br>AT3G61250|TGT > AGT, Cys217Ser<br>Polar hydrophilic > Polar<br>hydrophilic<br>Cline steepness: 0.3|Promotes transition<br>from vegetative growth<br>to flowering.|(Pastore et al., 2011)| |_LATE MERISTEM_<br>_IDENTITY2_ (_LMI2_) <br>*** TF type: R2R3**<br>**MYB**|MA_118448g0010/<br>AT3G61250|GGG > AGG, Gly140Arg<br>**Non-polar hydrophobic >**<br>**Polar hydrophilic**<br>Cline steepness: 0.3|GGG > AGG, Gly140Arg<br>**Non-polar hydrophobic >**<br>**Polar hydrophilic**<br>Cline steepness: 0.3|GGG > AGG, Gly140Arg<br>**Non-polar hydrophobic >**<br>**Polar hydrophilic**<br>Cline steepness: 0.3| |_FLOWERING_<br>_CONTROL LOCUS A_<br>(_FCA_)|MA_5232462g0010/<br>AT4G16280|CAA > AAA, Gln392Lys<br>Polar hydrophilic > Polar<br>hydrophilic<br>Cline steepness: 0.2|Promotes transition<br>from vegetative growth<br>to flowering.|(Macknight et al.,<br>1997)| |_TERMINAL_<br>_FLOWER 1_ (_TFL1_)|MA_178498g0010/<br>AT5G03840|GAT > GAA, Asp54Glu<br>Polar hydrophilic > Polar<br>hydrophilic<br>Cline steepness: 0.2|Suppresses floral<br>meristem, involved in<br>the regulation of<br>flowering time.|(Hanano and Goto,<br>2011)| 12 275 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 276 **Table 3** Weir and Cockerham F ST estimates for six populations (1654 Individuals) of Norway 277 spruce across Sweden involved in the detection of latitudinal clines (including 238 512 biallelic 278 missense SNPs). 279 |Population|S2|S3|S4|S5|S6| |---|---|---|---|---|---| |S1|0.00042993|0.0052606|0.016703|0.024725|0.029301| |S2||0.0030387|0.013321|0.021762|0.025045| |S3|||0.0062921|0.014399|0.015515| |S4||||0.0028874|0.0060752| |S5|||||0.0053678| 13 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 280 **Discussion** 281 _Weak neutral genetic differentiation contrasts with steep adaptive trait clines following_ 282 _postglacial recolonisation_ 283 284 Conifers originated approximately 300 million years ago and they have evolved over millions 285 of years to adapt to steep latitudinal and longitudinal environmental gradients (Williams, 2009). 286 The genetic composition of Norway spruce populations in Sweden reflects this evolutionary 287 history, which underwent post-glacial recolonisation by spruce migrating from two distinct 288 Last Glacial Maximum (LGM) refugia. Migration from the east via Russia and Finland 289 contributed to the recolonisation of Scandinavia, while Central European ancestry also 290 contributed to the genetic composition of southern and central Swedish populations (Chen et 291 al., 2019). 292 293 Norway spruce populations in Sweden appear to show weak population structure, with low 294 neutral genetic differentiation between populations (F ST ) owing to large population sizes and a 295 continuous distribution with few geographic barriers, allowing high pollen-mediated gene flow 296 (Chen et al., 2021, Eklöf et al., 2026). The Swedish Norway spruce population comprises two 297 distinct groups; the central and southern populations form one group and the other includes the 298 populations from northern Sweden (Chen et al., 2021). Weir and Cockerham F ST estimates for 299 six populations of Norway spruce across Sweden (Table 3) agree with several previous findings 300 regarding low F ST values suggesting low genetic differentiation (Chen et al., 2016, Ranade and 301 García-Gil, 2021, Ranade and García-Gil, 2023, Wang et al., 2020, Androsiuk et al., 2013). 302 303 A weak neutral population differentiation contrasts with a strong cline for adaptive traits (Q ST ). 304 The strongest evidence for local adaptation and optimal survival under given environmental 305 conditions in Norway spruce populations in Sweden is provided by the traits such as timing of 306 bud-burst and bud-set, as well as cold hardiness, height and drought tolerance (Clapham et al., 307 1998, Sogaard et al., 2008, Westin et al., 2000a, Westin et al., 2000b, Notivol et al., 2007). 308 Adaptive clines are the result of adaptation to temperature and daylength; in addition, light 309 quality also plays a crucial role (Ranade and García-Gil, 2021, Ranade and García-Gil, 2023, 310 Westin et al., 2000a). The northern populations of Norway spruce require far-red enriched light 311 to maintain growth, cease growth earlier, are more frost-resistant and disease-resilient than 312 southern populations, indicating adaptation to local climatic conditions (Ranade et al., 2022, 313 Ranade and García-Gil, 2021, Molmann et al., 2006, Westin et al., 2000b). 14 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 314 315 The genetic basis of the steep clines observed for adaptive traits are the result of clinal variation 316 in the DNA sequence and expression patterns of genes involved in phenology, lignin synthesis 317 and defence ultimately influencing trait variation along the cline (Ranade and García-Gil, 2021, 318 Ranade and García-Gil, 2023, Ranade and García-Gil, 2025, Ranade et al., 2022). The current 319 work provides further support for local adaptation in Norway spruce in genes regulating DNA 320 repair, defence response and flowering. Previously, the north-south genetic gradients in allele 321 frequency and gene expression have been detected in several genes including phytochromes 322 (central regulators of light pathway), genes involved in cell wall formation and defence, which 323 are proposed to be due to local environmental adaptation (Ranade and García-Gil, 2021, 324 Ranade and García-Gil, 2023, Ranade et al., 2022). In those cases, F ST values increase with 325 geographic distance, potentially reflecting reduced pollen-mediated gene flow after certain 326 distance and local adaptation to environmental conditions that favour different alleles. 327 328 _Latitudinal clines in the regulation of flowering_ 329 Reproduction is fundamental for species survival. Adaptation and genetic diversity plays a 330 crucial role in the process of reproduction. Flowering and seed production are the key events 331 in the reproductive cycle in plants. The flowering phase involves a shift from vegetative to 332 reproductive growth, initiating a reallocation of resources, often affecting the tree biomass, 333 with effects depending on the species and environmental conditions (Zhao et al., 2021). 334 Flowering depends on the climatic conditions; plants flower early or late depending mainly on 335 the light conditions (photoperiod), temperature and degree days. Recent studies in flowering 336 plants have reported that initiation of flowering correlates with latitude as the process is 337 governed by climatic gradients (Landoni et al., 2024). Ecotypic variation in flowering time 338 owing to sequence variations in flowering genes is associated with adaptation across natural 339 seasonal environments (Fournier-Level et al., 2022). Furthermore, epistasis can influence 340 flowering time following a latitudinal cline (Caicedo et al., 2004). However, in conifers this 341 area remains less explored from the molecular point of view. The current work may provide 342 the basis for understanding this complex network as we report gene variation in flowering 343 related genes that may contribute to difference in flowering time in different Norway spruce 344 populations; e.g. the northern Norway spruce populations flower early in the growing season 345 than the southern while the southern ones flower more frequently compared to the northern 346 populations (Danusevicius and Brazaitis, 2011, Nikkanen and Ruotsalainen, 2000). In the 347 current analysis, moderate to mild clinal variations in cSNPs were detected in floral activators 15 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 348 ( _VRN1_, _LMI2_, _FCA_ ) as well as repressors of flowering ( _JMJ27_, _TFL1_ ), along with genes that 349 regulate the development of the floral meristem in general ( _BAM1_ ) (Table 2). _VRN1,_ which 350 showed a moderate cline of steepness of 0.4, encodes a B3-type of TF essential for the 351 repression of FLC in vernalized in _Arabidopsis_ plants (King et al., 2013). Other target genes of 352 _VRN1_ includes genes that regulate frost tolerance (Deng et al., 2015). This is consistent with 353 the latitudinal temperature gradient in Sweden, where northern latitudes experience much 354 lower freezing temperatures than southern latitudes. SNP variation along the temperature 355 gradient might have resulted in populations adapted to the local temperature conditions. 356 357 Although the scope of the current work was limited only to missense mutations, it is worth 358 analysing the synonymous polymorphisms because they can alter RNA secondary structure. 359 Clinal variation was detected in a synonymous cSNP (771C>T) in _SQUAMOSA BINDING_ 360 _PROTEIN-LIKE1_ ( _SPL1_ ) gene that harbours the _acrocona_ mutant-specific SNP in its 361 miRNA156/ miRNA529 binding site due to which this mutant displays early cone setting 362 (Akhter et al., 2022). _SPL1_ is active in reproductive meristems while miRNA156/miRNA529 363 are present in high levels at juvenile plant stages and they prevent early flowering by 364 suppressing the _SPL1_ gene. The _acrocona_ mutant-specific SNP reduces 365 miRNA156/miRNA529-mediated repression, allowing early cone setting (Akhter et al., 2022). 366 A synonymous polymorphism can modulate microRNA(miRNA)-mRNA interactions and can 367 and alter the secondary RNA structure which may influence the accessibility of miRNA 368 binding sites. The 771C>T SNP from _SPL1_ displays a standard cline and it is present 309 bp 369 away from the miR529/miR156-binding site (Supplementary file2 – Figure S25). Although this 370 SNP is located too far (309 bp) to directly disrupt the binding site, it can still affect gene 371 regulation by changing the overall structure of the RNA strand and influence the availability 372 of miRNA-binding sites (Kertesz et al., 2007). In this context, the 771C>T SNP from _SPL1_, 373 may be involved in the regulation of flowering time. With the given scenario, it is noteworthy 374 that the data from the current investigation come from exome capture and hence do not cover 375 the entire gene. It would be strategic to sequence entire _SPL1_ gene from a sufficiently large 376 population along the latitudinal cline, to reveal other SNPs located in the vicinity of 377 miRNA156/ miRNA529-binding site, which would give information of one of the key aspects 378 regarding molecular basis of regulation of flowering time in Norway spruce. 379 380 The current analysis was performed using an exome capture dataset; therefore, the SNPs were 381 detected from a small sequence from the coding region of the gene. Although a synonymous 16 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 382 SNP does not change the encoded amino acid, yet it identifies a variable region. A missense 383 SNP positioned close to a synonymous SNP may have remained unidentified because the 384 exome capture probe was not designed to detect it. The neighbouring SNPs are often in linkage 385 disequilibrium and may show similar allele frequencies and association signals. In this context, 386 yet another synonymous SNP from _STYLISH1_ ( _STY1_ ) displayed a steep cline of 0.6 387 (Supplementary file2 – Figure S26); _STY1_ is a transcription factor that regulates floral organ 388 development and plays a role in controlling flowering time as well as in the maintenance of 389 apical meristem (Ståldal et al., 2012). Sequencing the entire gene will provide a more 390 comprehensive picture of local adaptation associated with the gene of interest. 391 392 Variants from several transcription factors involved in the regulation of flowering displayed 393 clinal variation. Few examples of key TFs include _VRN1_ (King et al., 2013), _LMI2_ (Pastore et 394 al., 2011), _AGAMOUS-LIKE 20_ ( _AGL20_ ), _APETALA 2 (AP2_ ) (Yant et al., 2010) and _LONG_ 395 _VEGETATIVE PHASE 1_ ( _LOV1_ ) (Yoo et al., 2007). These TFs have cline steepness values 396 ranging from 0.1 to 0.4 and belong to the B3, R2R3 MYB, MADS box, AP2/EREBP and NAC 397 domain transcription factor types, respectively (Supplementary file1 – Table S1). All these TFs 398 are key regulators of flowering and control the formation of floral organs and flowering 399 time. In addition to repression of flowering, _LOV1_ plays a central role in promoting 400 cold/freezing tolerance (Yoo et al., 2007) and it was found to be upregulated in response to FR 401 enriched light in the northern Norway spruce populations as compared to the southern ones 402 (Ranade and García-Gil, 2021). Here again it is worth noticing the steep temperature gradient 403 along the latitudes under consideration. AGL20 acts as a major floral activator and it promotes 404 the transition to flowering (Lee et al., 2000), while AP2 represses flowering; AP2 acts as a 405 repressor to inhibit AGL20 (Yant et al., 2010). Overall, regulation of flowering involves a 406 complex network of TFs and other genes involved in the formation of floral organs which are 407 interconnected. cSNPs variants that follow a latitudinal cline represent local adaptations to the 408 climatic clues and facilitate the tree to survive, thrive and reproduce at its best in its 409 environment. 410 411 _Association of variation in cSNPs with bud-set/bud-burst cline_ 412 Bud-set or growth cessation occurs in late summer/autumn, and bud-burst refers to growth 413 reactivation in spring. Both traits follow steep latitudinal clines in Norway spruce and depend 414 on climatic cues, namely photoperiod and temperature; they allow the tree to maximize its 415 growing season while minimising the risk of severe frost damage (Svystun et al., 2021, Ranade 17 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 416 and García-Gil, 2021). Bud-burst is governed mainly by temperature; the northern Norway 417 spruce populations flush earlier with the first signs of sustained warmth, while southern trees 418 result in later bud-burst that protects them against spring frost (Sogaard et al., 2008, Svystun et 419 al., 2021). Epigenetic memory plays a crucial role in Norway spruce growth rhythm, for 420 example, epigenetic memory of warm temperatures during embryogenesis leads to earlier bud 421 burst altering the expression of key phenological genes like _FLOWERING LOCUS_ 422 _T/TERMINAL FLOWER 1-_ like gene ( _PaFTL2_ ) (Carneros et al., 2017) _._ In Norway spruce, the 423 _Terminal Flower 1_ and _Terminal Flower 2_ refer to FLOWERING LOCUS T/TERMINAL 424 FLOWER 1-Like genes (abbreviated as _PaFTL1_ and _PaFTL2_ ) (Karlgren et al., 2013). _PaFTL1_ 425 and _PaFTL2_ are paralogous genes; their temporal and spatial expression largely complement 426 each other, suggesting their simultaneous role in regulating perennial growth in Norway spruce 427 (Karlgren et al., 2013). While _TFL1_ represses floral meristem development, _TFL2_ induces bud 428 set and growth cessation in _Arabidopsis_ (Larsson et al., 1998). Although the timing of bud-set 429 and bud-burst following a steep latitudinal cline has been well documented in Norway spruce, 430 the underlying molecular mechanisms are less explored owing to the complex multigenic 431 involvement. In the current analysis, we detected a moderate cline in a cSNP associated with 432 _TFL1_ or the _PaFTL1_, which may contribute to clinal variation of bud-set/bud-burst in Norway 433 spruce. 434 435 _Stress response and disease resistance_ 436 _RPL18C, ZDP, OXR1_ and _TPS02_ (Table 1) are defence-related genes whose cSNPs show steep 437 clines in allele frequency. _RPL18C_ plays a key role in the defence mechanism in eukaryotes 438 including humans (Dhar et al., 2006). Likewise, _OXR1_ protects the cells from oxidative stress 439 and mediates DNA repair in mammalian cells too (Matsui et al., 2020). Moreover, the tolerance 440 to oxidative stress may lead to an increase in plant biomass (Colombatti et al., 2019). 441 Interesting ecotypic variation has been observed in _Arabidopsis_ ; the Col-0 ecotype in 442 _Arabidopsis_ possesses a non-functional _TPS02_ due to a frame-shift mutation, whereas in the 443 Ws ecotype, it is a key functional gene without the mutation (Huang et al., 2010). In this 444 context, the variation in Norway spruce _TPS02_ may be linked to a similar phenomenon 445 conferring resistance to the northern populations (Ranade et al., 2022). Three cSNPs were 446 detected in _ZDP_, which is involved in DNA repair and defence; although the ZDP sequence 447 obtained from PlantGenIE was partial, it contained the conserved protein domain HAD_PNP 448 (Supplementary file2 – Figure S5) which was identified using the Conserved Domain Database 18 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 449 (CDD); the HAD_PNP domain plays a key role in both base excision repair and non 450 homologous end-joining DNA repair pathways (Marchler-Bauer et al., 2015). 451 452 cSNPs from several genes (Table 1, Supplementary file1 – Table S1) involved in stress response 453 and disease resistance followed a latitudinal cline including genes from various pathways 454 contributing to disease resistance like the lignin, phenylpropanoid, terpenoid and flavonoid 455 pathways (e.g. MYBs and peroxidases). Another example is MAP KINASE SUBSTRATE 1 456 (MKS1) which plays a key role in modulating plant innate immunity and regulating pathogen 457 defence mechanisms through salicylic acid and jasmonate pathways (Andreasson et al., 2005). 458 Several genes from the Leucine-Rich Repeat (LRR) family show clines in the cSNPs 459 (Supplementary file1 – Table S1); LRR family proteins are immune receptors involved in plant 460 defence (Hao et al., 2025). cSNPs from genes regulating drought stress responses, such as 461 aquaporin genes (Afzal et al., 2016) and genes involved in abscisic acid (ABA) signalling 462 (Sharma et al., 2023), display moderate clines. Although majority of these cSNPs displayed 463 mild clines, the respective genes involved may have a combined effect on the defence 464 mechanism, through complex networks taking part in the stress response. 465 466 _Genes contributing to “tree-ness” and tree growth in general_ 467 A recent comparative analysis of tree species proposed that unique genes defining a “woody 468 tree”, do not exist. Instead, trees have a genetic makeup similar to that of non-woody plants 469 and herbs. The study concluded that the trees organize and regulate these shared genes 470 differently which renders them the “tree-ness”. Few examples of such genes are expansins, 471 laccases, peroxidases and the MYB-related genes, which are essential for secondary cell wall 472 development leading to wood formation (Supplementary file1 – Table S1) (Ranade et al., 2022, 473 Ranade and García-Gil, 2025). Genes involved in plant growth in general, displayed 474 moderate/mild clines in their cSNPs; few examples include _SLOW GROWTH_ genes ( _SLO2_, 475 _SLO3_ ), _FCS LIKE ZINC FINGER 6_ ( _FLZ6_ ) and _FRUCTOSE-BISPHOSPHATE ALDOLASE 6_ 476 ( _FBA6_ ) (Hsieh et al., 2015, Jamsheer et al., 2018, Liu et al., 2025) (Supplementary file1 – Table 477 S1). 478 479 Light pathways modulate environmental signalling and are the key contributors towards 480 metabolic energy production controlling the overall tree growth. It has already been 481 documented that phytochromes, central regulators of light pathway show distinct clines in their 482 allele frequencies (Ranade and García-Gil, 2023). Another worth mentioning example from 19 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 483 this pathway is _LIGHT-HARVESTING COMPLEX B7_ ( _LHCB7_ ), a chlorophyll A-B binding 484 family protein that fine-tunes the rate of photosynthesis (Mei et al., 2025). A missense cSNP 485 (Thr125Ala) from _LHCB7_ displayed a trend cline, having a cline steepness of 0.4 486 (Supplementary file2, Figure S21). 487 488 _Significance of clinal variation for tree breeding_ 489 Clinal variation provides a useful basis for selecting forest reproductive material suited to 490 different growing conditions. In Norway spruce, traits such as cold hardiness, bud flush and 491 growth cessation vary with latitude, reflecting adaptation to local temperature and light 492 conditions. Linking these patterns to genetic variation can help identify material that combines 493 growth with frost hardiness and disease resistance and guide its deployment as the climate 494 changes. For example, northern Swedish populations generally show greater cold tolerance and 495 have shown stronger defence responses, whereas southern populations tend to grow faster. 496 Understanding the genetic basis of these differences can help breeders balance productivity and 497 resilience, identify candidate markers for genomic selection, and guide assisted migration. 498 499 However, transferring trees along a latitudinal cline cannot be guided by temperature alone. 500 Warming does not alter the daylength associated with a given latitude, and seasonal patterns of 501 light quality will not simply shift northwards with temperature. Although light quality can vary 502 with atmospheric and canopy conditions, its latitude-dependent seasonal patterns remain 503 relevant to adaptation. Trees transferred northwards may therefore encounter warmer 504 conditions that favour growth while responding inappropriately to local photoperiod and light 505 quality, with consequences for growth cessation, bud set and cold acclimation. Such 506 mismatches may also affect reproductive development, including the initiation and timing of 507 flowering and the synchronisation of pollen release and female receptivity, potentially 508 influencing seed production. Breeding and deployment strategies should therefore evaluate 509 genetic material that combines favourable growth and defence alleles with alleles supporting 510 vegetative and reproductive responses adapted to the light conditions at the site of deployment. 511 Clinal information can help identify candidate combinations and define seed-transfer limits, 512 reducing the risk of winter damage, poor growth or impaired reproductive performance when 513 southern or coastal material is planted at northern or high-elevation sites (Luoranen et al., 514 2022). 515 516 20 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 517 **Conclusions** 518 Our findings reveal pronounced clinal variation in genes associated with defence and 519 reproduction, against a background of weak neutral genetic differentiation in Norway spruce. 520 These genes provide candidates for understanding how populations adapt to contrasting 521 growing conditions. The results also highlight why assisted migration, and breeding strategies 522 should consider more than rising temperatures: daylength will remain unchanged, and seasonal 523 light conditions will not shift northwards with warming. Evaluating genetic material that 524 combines growth and defence with vegetative and reproductive responses suited to the 525 destination could help reduce maladaptation and support productive, resilient forests under a 526 changing climate. 21 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 527 **Acknowledgements** 528 This work was partially supported by the Wallenberg Initiatives in Forest Research 529 (WIFORCE) funded by the Knut and Alice Wallenberg Foundation (KAW 2020.0240). The 530 authors acknowledge the support from the support from Science for Life Laboratory 531 (SciLifeLab), the Swedish Foundation for Strategic Research (SSF), Bioinformatics Facility at 532 the Umeå Plant Science Centre, the Uppsala Multidisciplinary Center for Advanced 533 Computational Science at Uppsala University, and the PDC Center for High Performance 534 Computing at KTH Royal Institute of Technology for access to computational resources. 535 Computational resources were provided by the National Academic Infrastructure for 536 Supercomputing in Sweden (NAISS), funded by the Swedish Research Council. 537 538 **Availability of data** 539 The data that supports the findings of this study are available in the additional files of this 540 article. The exome capture raw reads and the RNA-seq data have been deposited in NCBI’s 541 sequence read archive (SRA) under accession number (PRJNA731384). 542 543 **Competing interests** 544 The authors declare no conflict of interest. 545 546 **Authors' contributions** 547 SSR: Conceptualization, Data curation, Formal analysis, Methodology, Software, 548 Visualization, Writing – original draft, Writing – review & editing; MRGG: Conceptualization, 549 Funding acquisition, Project administration, Resources, Supervision, Writing – review & 550 editing. 22 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764856; this version posted November 12, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764856) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 551 **References** 552 AFZAL, Z., HOWTON, T. C., SUN, Y. L. & MUKHTAR, M. S. 2016. 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