Malus domestica

We collected GBS data from a collection of 1995 accessions from the genus Malus from the US Department of Agriculture apple germplasm repository in Geneva, NY. The samples were processed with two different restriction enzymes (ApeKI, PstI/EcoT22I) in separate GBS libraries and were sequenced using Illumina Hi-Sequation 2000 technology. Genotypes were called using a custom GBS pipeline described in Gardner et al. (2014) (link). Briefly, 100-bp reads generated from both enzymes were aligned to the Malus domestica reference genome version 1.0 (Velasco et al. 2010 (link)) using the default parameters in BWA (Li and Durbin 2009 (link)). Genotypes were called using GATK (McKenna et al. 2010 (link)) with a minimum of eight reads supporting each genotype. The final genotype matrix was filtered to contain only samples from the domesticated apple, Malus domestica, and ≤20% missing data per SNP and per sample. SNPs with a minor allele frequency (MAF) of <0.01 were then discarded. Finally, the data were pruned to exclude clonal relationships: if two or more samples had IBD >0.9, they were considered clones and the sample with the least amount of missing data from the group was retained. This resulted in a dataset of 711 samples and 8404 SNPs.
To test the accuracy of our imputation method we created a “masked” dataset by setting 10,000 random genotypes to missing. This created “truth known” genotypes to which our imputed genotype calls were compared. We limited our testing to 10,000 masked genotypes, which represents 0.17% of the genotype matrix, in order to maintain a dataset with a reasonable amount of missing data while providing enough masked genotypes to be able to estimate imputation accuracy.
Biased allele frequency in imputed data has been shown to affect downstream analyses (Han et al. 2014 (link)). To determine how well each imputation method estimates allele frequencies, we filtered the genotype matrix to contain no missing data. This resulted in a matrix containing 1001 SNPs from 459 samples (Figure S2). We masked and then imputed 20% (91,952 genotypes) of the genotypes at random and compared the allele frequency estimates from the imputed data to the allele frequency estimates from the complete genotype matrix. As most imputation methods make use of other SNPs to aid imputation, we imputed using all 8404 SNPs in the dataset so as to provide more information to these methods. We then restrict our analysis to the 1001 complete SNPs.
We also tested the performance of our method on genome-wide SNP data from maize and grape. The maize data were downloaded from the International Maize and Wheat Improvement Center (Hearne et al. 2014 ). We reduced the data to biallelic SNPs with <20% missing data and a MAF >1% and then discarded samples with >20% missing data. This resulted in 43,696 SNPs from 4300 samples.
To generate the grape dataset we collected GBS data from a collection of diverse samples from the genus Vitis including commercial Vitis vinifera varieties, hybrids and wild accessions from the USDA grape germplasm collection. The samples were processed with two different restriction enzymes (HindIII/BfaI, HindIII/MseI) and were sequenced using Illumina Hi-Sequation 2000 technology. We then used the 12X grape reference genome (Jaillon et al. 2007 (link); Adam-Blondon et al. 2011 ) and the Tassel / BWA version 4 pipeline to generate a genotype matrix (Li and Durbin 2009 (link); Glaubitz et al. 2014 (link)). Default parameters were used at each stage except for the SNP output stage where we filtered for biallelic SNPs. We then removed any genotypes with fewer than eight supporting reads using vcftools (Danecek et al. 2011 (link)). Using PLINK (Purcell et al. 2007 (link)), we removed SNPs with >20% missing data before removing samples with >20% missing data. We then removed SNPs with excess heterozygosity (failed a Hardy−Weinberg equilibrium test with a p-value < 0.001) and finally SNPs with a MAF < 0.01. This created a dataset of 8506 SNPs and 77 samples.

A 0.97 kb region of the Arabidopsis CHS promoter (At5g13930) was amplified by PCR from the Arabidopsis ecotype Columbia using the primers RPH-179 and RPH-180, then digested with KpnI and NcoI and cloned into the MCS of pGreenII 0800-LUC. The 1.04 kb pea CHS-1a promoter [GenBank: X80007] was subcloned into pGreenII 0800-LUC as an EcoRI-NcoI fragment [28 ]. A 0.92 kb Petunia CHS-A promoter [GenBank: X14591] was amplified by PCR using primers RPH-332 and RPH-333 from a V26 genomic DNA, digested with KpnI and NcoI and cloned into pGreenII 0800-LUC. The 1.3 kb Apple CHS1 promoter [GenBank: DQ022678] was isolated from Malus domestica Royal Gala, using the Genome Walker kit (Clonetech) with gene specific primers RPH-198 and RPH-199, then cloned into pGEM T-easy (Promega) and subcloned as a SalI-NcoI fragment into pGreenII 0800-LUC. pwo-Polymerase (Roche) was used for all PCR amplifications and cloned genes were sequenced to confirm no sequence modifications were incorporated.

This study was carried out at the fruit demonstration station of Hebei Agricultural University, Shunping county, Hebei province (38.97N, 114.92E), China. The newly grafted (NG) axillary buds and 1-year-old (OY) saplings examined in this study were from ‘Odysso’ (red-fleshed apple fruits with high anthocyanin contents bred by Markus Kobelt in Switzerland) and ‘Tonami’ (Malus domestica ‘Tonami’) apple cultivars. The virus-free M. robusta rootstocks used for grafting were rapidly propagated in a tissue culture system. Viroid-infected ‘Odysso’ was obtained by grafting onto ‘Tonami’ apple trees infected with ASSVd. Axillary buds on the annual branches of all scions were collected at the Hebei Agricultural University nursery in early March. The ‘Odysso’ and ‘Tonami’ saplings infected with ASSVd (i.e., treatments) were compared with the virus-free control ‘Odysso’ and ‘Tonami’ saplings. The grafted saplings were transferred to 45 cm plastic pots. Ten saplings were used per treatment. All saplings were grown under consistent conditions.

This study was conducted in an apple orchard at Laishan district, Yantai City, Shandong Province, China (121°42′55′′E, 37°49′58′′N), during the fruit expansion stage in 2018 and 2019. Thirty ‘Yanfu3’/M26/Malus hupehensis Rehd. apple trees were used as experimental material in this study. Trees were planted in the year 2013 in rows spaced 1.5 m apart with 4 m between the rows and were trained as a slender spindle. The density of the experimental site was 111 apple trees per 667 m². The mean temperatures in August, September, and October were 27.3°C, 22.1°C, and 14.6°C in 2018 and 27.1°C, 21.8°C, and 14.3°C in 2019, respectively. The precipitation in August, September, and October was 126.6, 52.2, and 4.1 mm in 2018 and 124.2, 51.3, and 3.5 mm in 2019, respectively. The soil was brown loam, the pH of soil was 5.77, the soil organic matter content was 12.56 g/kg, and available potassium (K), available phosphorus (P), NO₃-N, and ${\text{NH}}_4^{+}-\text{N}$ were 211.23, 59.51, 45.73, and 29.31 mg/kg, respectively.

¹⁵N labeling was performed on 1st August (95 days after blooming). Group 1 of each treatment was supplemented with 20 g of ¹⁵N-urea and 100 g of common urea, which were mixed and solutioned with MgSO₄ and then fertilized to the soil. The whole plant was destructively sampled on 20th October (180 days after flowering), and then the indexes correlated with ¹⁵N were determined.
¹³C isotope labeling was performed on 17th October (177 days after flowering). Fans, a beaker with 8 g of Ba¹³CO₃ and reduced iron powder, and the labeled whole apple tree were placed into a labeling chamber, which was composed of 0.1-mm-thick Mylar plastic bags and bracket. The light intensity inside was 90% of the natural light intensity. The ¹³C isotope labeling was performed at 8.30 a.m.; the fan was turned on, and the labeling room was sealed. To maintain a suitable temperature (25°C–35°C), an appropriate amount of ice was placed in the labeling room. To maintain a suitable concentration of CO₂, we injected 1 ml hydrochloric acid into the labeling room by a syringe for every 30 min. The whole plant was destructively after 72 h (180 days after flowering), and then the indexes correlated with ¹³C were determined.