Axiom 2.0 assay manual

DNA samples from all the NAM lines used in this study were extracted from young leaves using Thermo Scientific GeneJET Plant Genomic DNA Purification Mini Kit. The DNA samples were checked for quality on 0.8% agarose gels and quantified on a Nanodrop 8000 Spectrophotometer (Thermo Scientific, Pittsburgh, PA). Affymetrix GeneTitan platform was used to genotype both NAM populations with the 58K SNP ‘Axiom_Arachis’ array (Clevenger et al., 2017; Pandey et al., 2017b). Initially, the target probes for 581 samples for NAM‐T and 496 samples for NAM‐F were prepared using a minimum of 20 μL DNA with a concentration 10 ng/μL. The samples were then amplified, fragmented and hybridized on the array chip followed by single‐base extension through DNA ligation and signal amplification according to the procedure explained in the Affymetrix Axiom® 2.0 Assay Manual (http://axiom_2_assay_auto_workflow_user_guide.pdf). The GeneTitan Multi‐Channel Instrument (Affymetrix, Santa Clara, CA, USA) was then used for staining and scanning the samples to derive the genotypic information for each line. The genotypic data for each line were generated and stored in the form of.CEL file format.

For all subjects, leaf tissues were sampled. Genomic DNA was extracted and hybridized on the Wheat660K SNP genotyping array by Compass Biotechnology Company (Beijing, China). The DNA samples were prepared, and the chip genotyping was performed on the Wheat660K SNP array according to the Affymetrix Axiom 2.0 Assay Manual Workflow protocol. DNA integrity was confirmed on agarose gels, and DNA quantity was measured spectrophotometrically. The Wheat660K chip contains 630517 markers (http://wheat.pw.usda.gov/GG2/index.shtml). Variant quality from the Wheat660K chip genotyping was initially assessed according to Affymetrix best practices. The 188 RILs and their parents were aslo assayed using the ‘Wheat PstI (TaqI) 2.3D’ DArT array (the medium density array) (http://www.triticarte.com.au/). The PCR-based markers were genotyped as described in our previous study^{3 (link)}.

Affymetrix GeneTitan^®platform was used to genotype “Reference Set” with the SNP array. Initially the target probes were prepared using each DNA sample having minimum quantity of 20 μL of good quality DNA and 10 ng/μL concentration. This procedure is explained in detail in Affymetrix Axiom^® 2.0 Assay Manual. These samples were then amplified, fragmented and hybridized on chip followed by single-base extension through DNA ligation and signal amplification. This procedure is explained in detail in Affymetrix Axiom^® 2.0 Assay Manual Target Prep Protocol QRC. The GeneTitan^® Multi-Channel Instrument was then used for staining and scanning the samples and the details are provided in (http://media.affymetrix.com/support/downloads/manuals/axiom_2_assay_auto_workflow_user_guide.pdf).

Pigeonpea genomic DNA was isolated from young seedlings using CTAB method³⁸ , quality checked by electrophoresis in 1% agarose gel and quantified using Nano drop spectrophotometer. For target probe preparation, 20 μl of genomic DNA with concentration of 10 ng/ul was used according to Affymetrix Axiom® 2.0 Assay Manual. The samples were amplified using Target Prep Protocol QSCB1 (P/N 702990), fragmented, hybridized on the chip followed by single-base extension through DNA ligation and signal amplification. Affymetrix GeneTitan® Multi-Channel Instrument was used for staining, washing and scanning of the chip signals as per the manufacturer’s protocol (http://media.affymetrix.com/support/downloads/manuals/axiom_2_assay_auto_workflow_user_guide.pdf). SNP allele calling was done using Axiom™ Analysis Suite version 2.0 using its three workflows i.e., Best Practices, Sample QC, Genotyping and Summary (http://media.affymetrix.com/support/downloads/manuals/axiom_analysis_suite_user_guide.pdf) on the Affymetrix Gene Titan.CEL files. The Axiom Analysis Suite requires stored library files to convert.CEL files into genotype calls. SNPs with low call rate across the samples were removed and only good quality SNPs with a DQC of >0.85 and call rates of >95% were retained for further analysis.

A high resolution analysis of background recovery was done on a set of eight selected BC₁F_3:4 and BC₂F_2:3 lines in comparison with the RP Mushk Budji and the DP DHMAS 70 Q 164-1b using ‘OsSNPnks’ 50 K Axiom® 2.0 SNP array⁵² . High quality genomic DNA for the assay was quantified using Nanodrop spectrophotometer and concentration was adjusted to 20 ng/µl with OD_260/280 values in the range of 1.8–2.0.
For target probe preparation, 20 μl of gDNA was used for each DNA sample at a concentration of 10 ng/μL (for a total 200 ng DNA in 20 μl) based on Affymetrix Axiom® 2.0 Assay Manual. DNA amplification, fragmentation, chip hybridization, DNA ligation and signal amplification were performed using the Affymetrix Axiom® 2.0 Assay Manual Target Prep Protocol QRC (P/N 702990). Staining and scanning were performed on the GeneTitan® Multi-Channel Instrument according to the manufacturer’s procedure (http://media.affymetrix.com). The assay included 50,051 high quality non-redundant SNPs mostly representing single-copy (SC) genes from whole rice genome with an average interval of 7.45 Kb between SNPs.

DNA from 352 RILs and both parents was extracted using the NucleoSpin Plant II kit (Macharey-Nigel, Duren, Germany). The DNA quality was analysed on 0.8% agarose gel, and concentration was measured using a NanoDrop 8000 spectrophotometer (Thermo Scientific). An Affymetrix GeneTitan® platform was used to genotype the RIL population with the 58K SNPs “Axiom_Arachis” array (Pandey et al., 2017b (link)). Initially, the target probes for 352 samples were used in at least 20 µL DNA, with a concentration of 10 ng/µL. The samples were then amplified, fragmented, and hybridized on the array chip, followed by single-base extension through DNA ligation and signal amplification, according to the procedure explained in the Affymetrix Axiom 2.0 Assay manual (axiom_2_assay_auto_workflow_user_guide.pdf) (Pandey et al., 2020a (link)). Axiome_Arachis is an SNP array developed for genotyping genetic populations in groundnut for trait mapping and association mapping (Pandey et al., 2017b (link)).