Identification of SNPs associated with magnesium and sodium uptake and the effect of their accumulation on micro and macro nutrient levels in Vitis vinifera

Macro and micro nutrient accumulation affects all stages of plant growth and development. When nutrient deficiencies or excesses occur, normal plant growth is altered resulting in symptoms such as leaf chlorosis, plant stunting or death. In grapes, few genomic regions associated with nutrient accumulation or deficiencies have been identified. Our study evaluated micro and macro nutrient concentrations in Vitis vinifera L. to identify associated SNPs using an association approach with genotype by sequencing data. Nutrient concentrations and foliar symptoms (leaf chlorosis and stunting) were compared among 249 F1 Vitis vinifera individuals in 2015 and 2016. Foliar symptoms were consistent (≥90%) between years and correlated with changes in nutrient concentrations of magnesium (r = 0.65 and r = 0.38 in 2015 and 2016, respectively), aluminum (r = 0.24 and r = 0.49), iron (r = 0.21 and r = 0.49), and sodium (r = 0.32 and r = 0.21). Single nucleotide polymorphisms associated with symptoms, sodium, and magnesium were detected on each chromosome with the exception of 5, 7 and 17 depending on the trait and genome used for analyses explaining up to 40% of the observed variation. Symptoms and magnesium concentration were primarily associated with SNPs on chromosome 3, while SNPs associated with increased sodium content were primarily found on chromosomes 11 and 18. Mean concentrations for each nutrient varied between years in the population between symptomatic and asymptomatic plants, but relative relationships were mostly consistent. These data suggest a complex relationship among foliar symptoms and micro and macro nutrients accumulating in grapevines.

240 with symptoms were observed for other nutrients, but were not consistent between years. When 241 comparing nutrient concentrations from 2015 to 2016, most nutrients had low to moderate (r = 242 0.2 -0.4) correlation, with the exception of copper (r= -0.0199) (Supplemental Table 3).
243 Nutrient ratios were examined between years for potential significant correlations with 244 symptoms. Most ratios did not show consistent differences in values between years for 245 symptomatic and non symptomatic vines (Supplemental Table 4).

247 Marker-trait associations
248 Linkage disequilibrium half-decay distance was estimated to be 100Kbp (Supplemental Figure   249 3). Genome-wide associations identified several chromosomes associated with differences in the  Table 5). In 2015, 6 SNPs were detected when aligned to the 254 PN40024 genome while 4 SNPs were detected when aligned to Thompson Seedless. No SNPs 255 associated with Mg accumulation were identified in 2016 with either genome at the P = 0.05 256 FDR level. Only 1 genic SNPs associated with Mg concentration was identified and was co-257 associated with SNPs identified for marginal leaf chlorosis and stunting (Supplemental Tables 6   258 and 7). In the Thompson Seedless genome, a small block of SNPs (S3_21825918 , 259 S3_21825925, and S3_21825966), spanning 48 bp, located in a ~ 7,000 bp intra genomic region, 260 explained ~ 18% of the variation associated with symptoms in 2015. A BLAST search of the 261 region did not identify any significant alignments with any genes (predicted, putative or known) 262 in Vitis or other species.

263
264 SNPs associated with Na concentration were detected on chromosomes 11, 12, 13, and 18 had 265 both negative and positive allelic effects ranging from 8 to 13% of the observed variation in the 266 Thompson Seedless genome. Three of the SNPs (S11_16152632, S18_25745134, and 267 S18_25745143) were detected in 2015 and 2016. None of the SNPs associated with Na 268 concentration in 2015 were located in predicted or known genes. Only a single SNP identified in 269 2016 (S11_11635675) was found in an annotated gene (Supplemental Table 6). In the PN40024 270 genome, 11 SNPs with positive allelic effects were detected across chromosomes 3, 11, 15, and Manuscript to be reviewed 271 17 in 2016. Individual SNPs explained 6 to 11% of the variation observed. Two of the SNPs on 272 chromosomes 3 and 11 were also associated with Na concentrations in 2015, but were not 273 significant when including adjusted for an FDR of 0.05. Using the PN40024 genome annotation, 274 8 genes were associated with Na accumulation. These included genes putatively involved 275 metabolism and transport. Four of the genes had no functional annotation ascribed (Table 5).
276 One gene, Vitvi11g01139, was associated with Na accumulation and annotated as a Clathrin 277 assembly protein, which is a class of proteins involved in macromolecule transportation. No   Table 6). When aligned to the PN40024 genome, a total of 29 and 55 SNPs in

304
305 No ion transport pathways were associated with symptom-associated SNPs based on the 306 Thompson Seedless annotation, however approximately 50% of the genes had putative catalytic 307 activity and 50% had binding activity (Supplemental Figure 5). None of the genes identified 308 across both years and associated with symptoms were putative transporters, but were instead 309 involved in processes such as oxidation, transcription, development, and stress response ( Table   310 6). When symptom-associated SNPs located in genes based on the PN40024 annotation were 311 evaluated for putative activity, stress response, transcription, growth and development, and 312 metabolic pathways were all represented similar to the Thompson Seedless genome. In addition, 313 SNPs were also detected in several genes related to sugar and nutrient transport. One SNP 314 associated with leaf stunting in both years and symptoms was associated with Calcium ion 315 binding (Vivi03g00243).

317
The majority of symptom-associated SNPs were found on chromosome 3 in both genomes, but 318 each genome has a unique coordinate system. Therefore, we performed a consolidated genome 319 analysis by mapping significant SNPs in Thompson seedless and their flanking regions to the 320 PN40024 genome in order to order SNP coordinates and look for overlap between references 321 ( Figure 4). When chromosome 3 assemblies were consolidated, a shared cluster of SNPs with 322 significant association with symptoms was observed ~7.5 Mb and a lesser cluster around ~15Mb.
323 When aligned to the Thompson Seedless genome, symptom-associated SNPs located within 324 genes shared across years were predominantly found on chromosome 3 with an additional SNP 325 located on chromosome 10 ( Table 6, Supplemental Table 6). Significant single year SNPs, 326 including those associated in genes, were identified on chromosomes 1, 2, 3, 4, 10, 11, 12, 16, 327 and 18 (Supplemental Table 5). When symptoms were combined, 28 SNPs were shared across 328 both years, and 29 were only identified in a single year (Supplemental Table 5). When aligned to 329 the PN40024 genome, significant SNPs were detected on chromosomes 3, 6, 11, 16, and 19.